Protein kinase required for RAS signal transduction

ABSTRACT

The kinase suppressor of Ras (Ksr), a novel protein kinase involved in the regulation of cell growth and differentiation, provides an important target for therapeutic intervention. The subject compositions also include nucleic acids which encode a Ksr kinase, and hybridization probes and primers capable of hybridizing with a Ksr gene. Such probes are used to identify mutant Ksr alleles associated with disease. The invention includes methods, including phosphorylation and binding assays, for screening chemical libraries for lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated Ksr activity or Ksr-dependent signal transduction.

This is a division of application Ser. No.08/571,758 filed Dec. 13, 1995, now U.S. Pat. No. 5,700,675.

The research carried out in the subject application was supported in part by grants from the National Institutes of Health. The government may have rights in any patent issuing on this application.

INTRODUCTION

1. Field of the Invention

The field of the invention is a protein kinase required for Ras signal transduction and its use in pharmaceutical screens.

2. Background

Ras plays a crucial role in diverse cellular processes, such as proliferation and differentiation, where it functions as a nodal point transmitting signals originating from receptor tyrosine kinases (RTKs) to a variety of effector molecules (reviewed in McCormick, 1994a; van der Geer et al., 1994; Burgering and Bos, 1995). Ras activation, which involves a switch from an inactive GDP-bound to an active GTP-bound state, is promoted by a guanine nucleotide-exchange factor. Upon RTK activation, the exchange factor is recruited by an SH2/SH3 domain-containing adaptor molecule to the RTK at the plasma membrane where it can contact and activate Ras. GTP-bound Ras then transmits the signal to downstream effector molecules.

The protein serine/threonine kinase Raf has been identified as a major effector of Ras (reviewed in Daum et al., 1994; McCormick, 1994b). Upon Ras activation, Raf is recruited to the plasma membrane by a direct interaction with Ras, where it is subsequently activated by an unknown mechanism. Raf activation initiates an evolutionarily conserved pathway involving two other kinases, MEK (MAPK Kinase) and MAPK (Mitogen-Activated Protein Kinase) that convey signals to the nucleus through a directional series of activating phosphorylations (reviewed in Marshall 1994). Although this model for Ras-dependent signal transduction is well-supported, there are still major issues that remain poorly understood. One of them is the mechanism by which Raf is activated. Recent evidence suggests that once recruited to the plasma membrane Raf is activated by phosphorylation (Dent and Sturgill, 1994; Dent et al., 1995). However, a candidate kinase(s) has yet to be identified. Another unresolved issue is the nature of other Ras effectors as well as the pathways they control. Although Raf is clearly a major Ras target, it can not account for all of the cellular responses mediated by Ras (for example see White et al., 1995).

Ectopic expression of an activated Ras1 allele, Ras1^(V12), in the developing Drosophila eye transforms non-neuronal cone cells into R7 photoreceptor cells (Fortini et al., 1992). Similar results are obtained by expression of an activated Drosophila Raf allele, D-Raf^(Tor4021) (Dickson et al., 1992). We carried out a genetic screen designed to isolate mutations that modify the signaling efficiency of Ras1^(V12). Most mutations that decreased the signaling efficiency of Ras1^(V12) also decreased the efficiency of D-Raf^(Torso4021) signaling. However, two groups of mutations were identified that did not alter D-Raf^(Torso4021) signaling. We disclose here the characterization of their respective loci. The Suppressor of Ras1 2--2 (SR2-2) locus encodes a protein homologous to the catalytic subunit of the prenylation enzyme type I geranylgeranyl transferase. We have renamed this locus βGGT-I. The second locus, SR3-1, encodes a novel protein kinase distantly related to Raf kinase members. Based on its sequence and the ability of mutants to reduce Ras1-mediated signaling, we renamed this locus kinase suppressor of ras (ksr). In addition to its function in the Sevenless RTK pathway, we show that ksr is also required for signaling by the Torso RTK We have isolated mouse and human homologs of ksr. Together, these data indicate that Ksr is an evolutionarily conserved component of the Ras signaling pathway. As such, the human Ksr provides an important target for pharmaceutical intervention.

Relevant Literature

Recent reports on Raf activation include Dent and Sturgill 1994; Dent et al., 1995; White et al., 1995, Yao et al, 1995; and a recent review by Marshall, 1994.

SUMMARY OF THE INVENTION

The invention provides methods and compositions relating to a novel protein kinase involved in the regulation of cell growth and differentiation: kinase suppressor of Ras (Ksr). As such, the kinase provides an important target for therapeutic intervention. The subject compositions also include nucleic acids which encode a Ksr kinase, and hybridization probes and primers capable of hybridizing with a Ksr gene. Such probes are used to identify mutant Ksr alleles associated with disease.

The invention includes methods for screening chemical libraries for lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated Ksr activity or Ksr-dependent signal transduction. In one embodiment, the methods involve (1) forming a mixture comprising a Ksr, a natural intracellular Ksr substrate or binding target such as the 14-3-3 gene product, and a candidate pharmacological agent; (2) incubating the mixture under conditions whereby, but for the presence of said candidate pharmacological agent, said Ksr selectively phosphorylates said substrate or binds said binding target at a control rate; and (3) detecting the presence or absence of a change in the specific phosphorylation of said substrate by said Ksr or phosphorylation or binding of said Ksr to said binding target, wherein such a change indicates that said candidate pharmacological agent is a lead compound for a pharmacological agent capable of modulating Ksr function.

DETAILED DESCRIPTION OF THE INVENTION

A Drosophila melanogaster, a Drosophila virilis, a murine and a human ksr encoding sequence are set out in SEQ ID NO: 1, 3, 5 and 7, respectively. A Drosophila melanogaster, a Drosophila virilis, a murine and a human ksr protein sequence are set out in SEQ ID NO: 2, 4, 6 and 8, respectively. Ksr proteins necessarily include a disclosed ksr kinase domain. Hence, Ksr proteins include deletion mutants of natural ksr proteins retaining the ksr kinase domain.

Natural nucleic acids encoding ksr proteins are readily isolated from cDNA libraries with PCR primers and hybridization probes containing portions of the nucleic acid sequence of SEQ ID NO: 1, 3, 5 and 7. Preferred ksr nucleic acids are capable of hybridizing with one of these sequences under low stringency conditions defined by a hybridization buffer consisting essentially of 1% Bovine Serum Albumin (BSA); 500 mM sodium phosphate (NaPO₄); 1 mM EDTA; 7% SDS at a temperature of 42° C. and a wash buffer consisting essentially of 2X SSC (600 mM NaCl; 60 mM Na Citrate); 0.1% SDS at 50° C.; more preferably under low stringency conditions defined by a hybridization buffer consisting essentially of 1% Bovine Serum Albumin (BSA); 500 mM sodium phosphate (NaPO4); 15% formamide; 1 mM EDTA; 7% SDS at a temperature of 50° C. and a wash buffer consisting essentially of 1X SSC (300 mM NaCl; 30 mM Na Citrate); 0.1% SDS at 50° C.; most preferably under low stringency conditions defined by a hybridization buffer consisting essentially of 1% Bovine Serum Albumin (BSA); 200 mM sodium phosphate (NaPO4); 15% formamide; 1 mM EDTA; 7% SDS at a temperature of 50° C. and a wash buffer consisting essentially of 0.5X SSC (150 mM NaCl; 15 mM Na Citrate); 0.1% SDS at 65° C.

The subject nucleic acids are recombinant, meaning they comprise a sequence joined to a nucleotide other than that to which sequence is naturally joined and isolated from a natural environment. The nucleic acids may be part of Ksr-expression vectors and may be incorporated into cells for expression and screening, transgenic animals for functional studies (e.g. the efficacy of candidate drugs for disease associated with expression of a Ksr), etc. These nucleic acids find a wide variety of applications including use as templates for transcription, hybridization probes, PCR primers, therapeutic nucleic acids, etc.; use in detecting the presence of Ksr genes and gene transcripts, in detecting or amplifying nucleic acids encoding additional Ksr homologs and structural analogs, and in gene therapy applications, e.g. using antisense nucleic acids or ribozymes comprising the disclosed Ksr sequences or their complements or reverse complements.

The invention also provides Ksr-specific binding reagents such as antibodies. Such reagents find a wide variety of application in biomedical research and diagnostics. For example, antibodies specific for mutant Ksr allele-products are used to identify mutant phenotypes associated with pathogenesis. Methods for making allele-specific antibodies are known in the art. For example, an mKsr-specific antibody was generated by immunizing mice with a unique N-terminal mKsr peptide (residues 118-249) GST fusion.

The invention provides efficient methods of identifying pharmacological agents or lead compounds for agents active at the level of a Ksr modulatable cellular function, particularly Ksr mediated signal transduction. For example, we have found that a binding complex comprising Ksr, 14-3-3 and Raf exists in stimulated cells; modulators of the stability of this complex effect signal transduction. Generally, the screening methods involve assaying for compounds which interfere with a Ksr activity such as kinase activity or target binding. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development. Target therapeutic indications are limited only in that the target cellular function be subject to modulation, usually inhibition, by disruption of the formation of a complex comprising Ksr and one or more natural Ksr intracellular binding targets including substrates or otherwise modulating Ksr kinase activity. Target indications may include infection, genetic disease, cell growth and regulatory or immunologic dysfunction, such as neoplasia, inflammation, hypersensitivity, etc.

A wide variety of assays for binding agents are provided including labeled in vitro kinase assays, protein-protein binding assays, immunoassays, cell based assays, etc. The Ksr compositions used in the methods are recombinantly produced from nucleic acids having the disclosed Ksr nucleotide sequences. The Ksr may be part of a fusion product with another peptide or polypeptide, e.g. a polypeptide that is capable of providing or enhancing protein-protein binding, stability under assay conditions (e.g. a tag for detection or anchoring), etc.

The assay mixtures comprise one or more natural intracellular Ksr binding targets including substrates, such as the 14-3-3 gene product, or, in the case of an autophosphorylation assay, the Ksr itself can function as the binding target. A Ksr-derived pseudosubstrate may be used or modified (e.g. A to S/T substitutions) to generate effective substrates for use in the subject kinase assays as can synthetic peptides or other protein substrates. Generally, Ksr-specificity of the binding agent is shown by kinase activity (i.e. the agent demonstrates activity of an Ksr substrate, agonist, antagonist, etc.) or binding equilibrium constants (usually at least about 10⁶ M⁻¹, preferably at least about 10⁸ M⁻¹, more preferably at least about 10⁹ M⁻¹). A wide variety of cell-based and cell-free assays may be used to demonstrate Ksr-specific binding; preferred are rapid in vitro, cell-free assays such as mediating or inhibiting Ksr-protein binding, phosphorylation assays, immunoassays, etc.

The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which may be used to facilitate optimal binding and/or reduce non-specific or background interactions, etc. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

In a preferred in vitro, binding assay, a mixture of a protein comprising at least one of the conserved Ksr domains, including CA1, CA2, CA3, CA4 and the kinase domain (see Table 1), one or more binding targets or substrates and the candidate agent is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the Ksr specifically binds the cellular binding target at a first binding affinity or phosphorylates the substrate at a first rate. After incubation, a second binding affinity or rate is detected. Detection may be effected in any convenient way. For cell-free binding assays, one of the components usually comprises or is coupled to a label. The label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, an enzyme, etc. A variety of methods may be used to detect the label depending on the nature of the label and other assay components. For example, the label may be detected bound to the solid substrate or a portion of the bound complex containing the label may be separated from the solid substrate, and thereafter the label detected.

The following experiments and examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Mutations in the SR2-2 and SR3-1 loci suppress the eye phenotype of activated Ras1 but not that of activated D-Raf.

Ectopic expression of activated Ras1 (Ras1^(V12)) under control of sevenless (sev) promoter/enhancer sequences (sev-Ras1^(V12)) transforms cone cells into R7 photoreceptor cells (Fortini et al., 1992). These extra R7 cells disorganize the ommatidial array, which causes a roughening of the external eye surface. The severity of eye roughness appears proportional to the strength of Ras1^(V12) -mediated signaling since two copies of the transgene produce a much more disrupted eye than one copy. We took advantage of this sensitized system to conduct a screen for mutations that reduce (suppressors) or increase (enhancers) the degree of eye roughness. We reasoned that a two-fold reduction in the dose of a gene (by mutating one of its two copies) that functions downstream of Ras1 should dominantly alter signaling strength which in turn should visibly modify the roughness of the eye. Based on this assumption, we screened ˜200,000 EMS- and ˜650,000 X-ray-mutagenized progeny for dominant modifiers of the Ras1^(V12) -mediated rough eye phenotype. 18 complementation groups of suppressors with multiple alleles and 13 complementation groups of enhancers of sev-Ras1^(V12) were isolated.

To characterize further the various groups of suppressors, we tested their ability to suppress dominantly the extra R7 cell phenotype caused by overexpression of an activated Drosophila Raf allele (sE-Raf^(Tor4021)). Since Raf functions directly downstream of Ras, we expected most of our suppressor groups to modify similarly the sE-Raf^(Tor4021) phenotype. Interestingly, two recessive lethal suppressor groups, SR2-2 and SR3-1 did not reduce the number of extra R7 cells produced by D-Raf^(Tor4021) expression. Scanning electron micrographs of adult eyes illustrate the suppressor phenotypes of one SR3-1 allele. Similar results were obtained with multiple SR2-2 and SR3-1 alleles. We also monitored the suppression of extra R7 cells by counting the number of R7 photoreceptors in cross-sections of adult fly retinae. In wild-type there is one R7 cell per ommatidium, whereas in sev-Ras1^(V12) /+ flies we observed 2.3 (n=437) R7 cells per ommatidium. This number was reduced to 1.2 (n=481) R7 cells per ommatidium in sev-Ras1^(V12) /+; SR3-1^(S-638/) + flies. In sE-Raf^(Tor4021) /+ flies, 2.3 (n=302) R7 cells per ommatidium were observed. However, this number remained at 2.3 (n=474) in sE-Raf^(Tor4021) /+; SR3-1^(S-638) /+ flies reflecting the inability of SR3-1 mutations to alter sE-Raf^(Tor4021) signaling strength.

Targeting of Ras1^(V12) to the plasma membrane by myristylation distinguishes SR2-2 from SR3-1.

Prenylation of the C-terminal CAAX box (C=cysteine, A=aliphatic residue, X=any amino acid) is the major post-translational modification specific to all Ras-like GTPases. When the residue at position "X" is a leucine, as in Ras1, a geranylgeranyl group is added by a type I geranylgeranyl transferase. The addition of this lipidic moiety is required to attach Ras to the plasma membrane (reviewed in Glomset and Farnsworth, 1994). Deletion of the CAAX box abolishes Ras function (Willumsen et al., 1984; Kato et al., 1992), however its activity can be restored if it is brought to the membrane by another localization signal, such as a myristyl group (Buss et al., 1989).

One possibility to account for the ability of a mutant to suppress sev-Ras1^(V12) but not sE-Raf^(Tor4021) is that the locus encodes an enzyme that is required for the membrane localization of Ras1. Consequently, mutations in this locus would not affect D-Raf^(Tor4021). To directly test this possibility, we asked if SR2-2 or SR3-1 alleles could suppress activated Ras1 if it is targeted to the membrane by an alternative mechanism. We targeted Ras1^(V12) to the membrane by fusing the first 90 amino acids of Drosophila Src kinase (D-Src; Simon et al., 1985), which contains a myristylation signal, to Ras1^(V12) deleted of its CAAX box (sev-Src90Ras1^(V12).increment.CAAX). While the CAAX box-deleted Ras1^(V12) is inactive, Src90Ras1 produces the same phenotype as Ras1^(V12) ; that is, it generates extra R7 cells and disrupts the ommatidial array.

We crossed sev-Src90Ras1^(V12).increment.CAAX flies to SR2-2 and SR3-1 alleles and analyzed the rough eye phenotype. SR2-2^(S-2110) did not suppress the rough eye phenotype while SR3-1^(S-638) suppressed the rough eye phenotype and the production of extra R7 cells. These observations indicate that SR2-2 is involved in prenylation of Ras1 while SR3-1 encodes a component of the Ras1 pathway that is not involved in the process of Ras1 membrane localization.

The SR2-2 locus encodes the Drosophila homolog of the β-subunit of type I geranylgeranyl transferase.

The SR2-2 locus was meiotically mapped to 2-15 (cytological position 25B-C), based on the ability of different mutant alleles to suppress sev-Ras1^(V12). One of the seven recessive lethal SR2-2 alleles recovered contains an X-ray-induced inversion (SR2-2^(S-2126)) with a breakpoint at 25B4-6. Genomic DNA spanning this breakpoint was isolated and used to screen a Drosophila eye-antennal imaginal disc cDNA library (see Experimental Procedures). A single class of cDNAs (ranging in size from 0.8 to 1.6 kb) defining a transcription unit disrupted by the inversion present in SR2-2^(S-2126), was identified and characterized. Conceptual translation of the longest open reading frame (ORF) defined by these cDNAs predicts a protein of 395 amino acids. Determination of the gene structure by sequencing the corresponding genomic region revealed four exons with the first in-frame methionine located at the beginning of the second exon. The SR2-2^(S-2126) inversion breakpoint maps to the 5'-end of the transcript. Further confirmation that this ORF corresponds to the SR2-2 gene, was provided by sequence analysis of two other mutant alleles, SR2-2^(S-483) and SR2-2^(S-2554), both of which have small deletions that remove the first exon and part of the 5' regulatory sequences. A search of the current protein databases with this ORF indicated that the SR2-2 gene encodes the Drosophila homolog of the catalytic β-subunit of type I geranylgeranyl transferase (BGGT-I) (Marshall, 1993). Sequence alignment with the human and the yeast S. pombe βGGT-I proteins shows a high degree of evolutionary conservation. The human sequence is 44% identical (69% similar) to the Drosophila sequence throughout the entire ORF while the yeast sequence is 36% identical (57% similar) to the Drosophila protein. We therefore renamed this locus, βGGT-I.

The SR3-1 locus encodes a novel protein kinase.

The ability of SR3-1 mutant alleles to suppress the sev-Ras1^(V12) phenotype was meiotically mapped to 3-47.5, which corresponds to a region near the chromocenter of the third chromosome. The map position was further refined by showing that SR3-1 meiotically maps between two P-elements inserted at 82F8-10 and 83A5-6, respectively. X-ray-induced chromosomal deletions were generated by selecting w⁻ revertants of one of the P-element insertions. One such deletion, Df(3R)e1025-14, which removes the chromosomal region from 82F8-10 to 83A1-3, complemented the SR3-1-associated lethality. Taken together, these results indicated that the SR3-1 locus lies between 83A1-3, the distal breakpoint of Df(3R)e1025-14, and 83A5-6, the insertion site of P w⁺ !5E2.

Five overlapping cosmids which cover this chromosomal region were recovered by chromosome walking. To identify restriction site polymorphisms that might have been induced in the SR3-1 alleles, these cosmids were used to probe genomic DNA blots prepared from 9 independent X-ray-induced SR3-1 alleles. Cosmid III revealed polymorphisms in a BamHI restriction digest of two alleles, SR3-1^(S-69) and SR3- 1^(S-511). No other cosmid revealed polymorphisms in the 9 tested alleles. A 7 kb SacII genomic fragment which spans the polymorphic BamHI fragments was introduced into the germline by P-element-mediated transformation. This genomic fragment, tested in transgenic flies, rescued both the lethality and the sev-Ras1^(V12) -suppression ability of three independent SR3-1 alleles. A single class of cDNAs that was totally encoded by the 7kb genomic fragment was identified by screening a Drosophila eye-antennal imaginal disc CDNA library and sequenced. The longest cDNA clone represents a transcript of 3.6 kb which is close to the size of a full-length transcript since RNA blot analysis identified a single band of similar size. Sequence analysis of the genomic region revealed that this transcript is encoded by a single exon. Conceptual translation of the longest ORF predicts a protein of 966 amino acids. The presence of an in-frame stop codon upstream of the predicted initiating methionine indicates that this cDNA contains the complete ORF.

A search of current protein databases indicated that SR3-1 encodes a novel protein kinase. The putative catalytic domain, which is C-terminal, contains the characteristic eleven conserved sub-domains found in eukaryotic kinases (Hardie and Hanks, 1995) and is preceded by a long N-terminal region with three distinctive features: a cysteine-rich domain similar to those found in Protein Kinase C isozymes (Hubbard et al., 1991) and Raf kinases (Bruder et al., 1992); four sequences that match the consensus phosphorylation site (PXS/TP) for MAPK (Marshall 1994); and a block of amino acids rich in serines and threonines followed by a conserved motif (FXFPXXS/T) that resembles the sequence around the Conserved Region 2 (CR2) domain of Raf kinases (Heidecker et al., 1992). Since the SR3-1 locus encodes a putative protein kinase and mutant alleles were isolated as suppressors of sev-Ras1^(V12), we renamed this locus kinase suppressor of ras (ksr).

Further confirmation that this gene corresponds to the ksr (SR3-1) locus was provided by sequencing three ksr alleles which revealed mutations disrupting the Ksr ORF (Table 1).

    TABLE 1                                                                           - Sequence comparison of the Ksr kinases.                                       ##STR1##                                                                        ##STR2##                                                                        ##STR3##                                                                        ##STR4##                                                                        ##STR5##                                                                        ##STR6##                                                                        ##STR7##                                                                        ##STR8##                                                                        ##STR9##                                                                        ##STR10##                                                                       ##STR11##                                                                       ##STR12##                                                                       ##STR13##                                                                       ##STR14##                                                                       ##STR15##                                                                       ##STR16##                                                                       ##STR17##                                                                       ##STR18##                                                                

Table 1 provides a detailed comparison of the predicted amino acid sequence of Ksr kinases. Conceptual translation of the open reading frame from the longest D. melanogaster (Dm) Ksr cDNA is shown. The positions of mutations in three ksr alleles are indicated: S-548 is a 4 bp X-ray-induced mutation affecting two consecutive codons (CTG-CGA to AGT-GGA). S-638 is an EMS-induced allele that has two separate point mutations changing a GCC codon to GTC and GCG codon to ACG. S-721 is a frameshift mutation due to a 10 bp duplication from adjacent sequences within the codon for asparagine-727. Also shown in the alignment are the conceptual translations of the open reading frames for the Ksr genes from other species: the D. virilis (Dv) Ksr sequence was derived from genomic DNA, the mouse (m) Ksr-1 from a 4 kb cDNA, and the human (h) Ksr-1, deduced from three overlapping cDNA clones (the N-terminal two residues were absent from these clones so the numbering begins with the third residue). The human Ksr is present as one or more of a plurality of alternatively spliced forms, exemplified by Ksr' in the following sequence listing. The amino acid sequences (and their respective positions) for the cysteine-rich regions and the kinase domains of Drosophila (D-Raf) and human (h c-Raf) (Genbank accession number: X07181 and X03484, respectively) are presented. Residues identical to Dm Ksr are lower case. In the N-terminus of the Ksr kinases four Conserved Areas (CA1 to CA4) are boxed. CA1 is a novel domain present only in the Ksr kinases. CA2 is a proline-rich stretch that may represent an SH3-binding site (Alexandropoulos et al., 1995). CA3 is a cysteine-rich stretch, simlar to a domain found in multiple signaling molecules. This conserved sequence is also part of the CR1 domain found in Raf kinases (Bruder et al., 1992). CA4 is a long serine/threonine-rich stretch followed by a conserved motif (indicated by a dashed line). This domain resembles the region around the CR2 domain of Raf kinases (Heidecker et al., 1992). The four short thick lines overlying the sequences indicate potential sites of phosphorylation by MAPK (PXS/TP) found in Dm Ksr. The eleven conserved sub-domains characteristic of protein kinases are indicated by roman numerals below their approximate positions.

ksr^(S-638) has two single amino acids changes: alanine-696 to valine and alanine-703 to threonine. The latter substitution alters a highly conserved residue within kinase sub-domain II (Hanks et al., 1988). ksr^(S-721) contains a 10 bp insertion in the codon for asparagine-727 within kinase sub-domain III creating a frameshift mutation that truncates the protein at kinase sub-domain III. ksr^(S-548) has a four base pair substitution that changes two consecutive amino acids in the N-terminus of the protein: leucine-50 and arginine-51 to glycine and serine, respectively. Unlike the 16 alleles recovered in the screen which were recessive lethal, ksr^(S-548) produces sub-viable flies which have rough eyes (see below), indicating that it is a weak loss-of-function mutation.

Identification of Ksr homologs in other species defines a novel class of kinases related to Raf kinases.

As a first attempt to determine functionally important domains that comprise the Ksr kinase, we searched for homologs from other species. First, we isolated the complete coding region of ksr from a Drosophila virilis genomic library by low-stringency hybridization (see Experimental Procedures). The D. virilis genomic sequence revealed a single uninterrupted ORF predicting a protein of 1003 amino acids (Table 1). The D. virilis and D. melanogaster Ksr proteins are 96% identical within the kinase domain while the N-terminal region is more divergent (69% identity), although islands of high conservation are present (see Table 1).

A search of translated nucleotide databases (using the TBLASTN program; Altschul et al., 1990) identified a partial ORF derived from a mouse DNA sequence with significant blocks of similarity to the N-terminus of Ksr. This sequence, named hb, had been isolated by Nehls et al. (1994) as part of an exon-trapping strategy to establish the transcription map of a 1 Mb region around the mouse NF1 locus. To determine if the full-length hb transcript also contains a kinase domain related to Ksr, we screened a cDNA library derived from a mouse PCC4 teratocarcinoma cell line with a probe corresponding to the hb sequence (see Experimental Procedures). A 4 kb cDNA clone was isolated and encodes a protein of 873 amino acids that contains a kinase domain highly related to the Ksr kinase domain (51% identity/74% similarity; Table 1). In addition, a human fetal brain cDNA library was screened at low-stringency with the same hb probe (see Experimental Procedures). Thirteen independent cDNA clones were purified and sequenced. They represent partial transcripts ranging in size from 0.6 to 3 kb. Interestingly, they define at least three classes of N-terminal splicing variants. The predicted protein sequence derived from overlapping human cDNA clones is shown in Table 1. With the exception of the first divergent 23 amino acids, which probably represents an alternative exon, human Ksr- 1 (hKsr-1) is nearly identical to mouse Ksr-1 (mKsr-1; 95% identity/99% similarity). Subsequent to this analysis, two human Expressed Sequence Tags (GenBank accession numbers: R27352 and R27353) have been reported that correspond to regions of the hKsr kinase domain.

Comparison of mammalian and Drosophila Ksr sequences showed similarity throughout the kinase domain as well as at various locations within the N-terminal region (Table 1). Sequence conservation is obvious within all sub-domains of the kinase domain. Two interesting features are present within sub-domains VIb and VIII. HRDL(K/R/A)XXN (D and N are invariant residues) is the consensus sequence corresponding to sub-domain VIb for the majority of known kinases (Hardie and Hanks, 1995). Instead of an arginine at the second position, a lysine is present for the Ksr homologs which distinguishes them from most other kinases. In addition, the amino acids N-terminal to the APE motif in sub-domain Vm, which have been implicated in substrate recognition specificity, (Hardie and Hanks, 1995) are well-conserved between the Ksr kinases of different species, but differ from those of all other kinases. One peculiarity is found in sub-domain II of the two mammalian proteins. This sub-domain has an invariant lysine residue involved in the phospho-transfer reaction that is conserved in all kinases identified thus far (Hardie and Hanks, 1995), however, both mammalian sequences have an arginine at this position (Table 1). It has been shown that mutagenesis of this lysine residue to any other residue, including arginine, abolishes catalytic function in several kinases (Hanks et al., 1988). However, the sequence conservation between the mouse and the human kinase domains indicates that these enzymes are functional.

Sub-domains VIb and VIII also contain conserved residues that often correlate with hydroxy amino acid recognition (Hanks et al., 1988). For instance, HRDLKXXN (VIb) and T/SXXY/F (VIII) motifs are indicative of Ser/Thr-kinases while HRDLRIAXA/RN'(VIb) and PXXW (VIII) motifs are associated with Tyr-kinases. Based solely on these conserved residues it is not clear to which class Ksr kinases belong (Table 1). Indeed, for sub-domain VIb, the Drosophila sequences have an arginine residue at the critical position (like a Tyr-kinase), while the two mammalian sequences have a lysine residue (like a Ser/Thr-kinase). The sub-domain VIII motif for all the Ksr members is WXXY, which differs from that found in all other kinases.

In the N-terminal region, four Conserved Areas (CA1 to CA4) can be recognized (Table 1). CA1 is a stretch of 40 amino acids located at the very N-terminus of Ksr kinases and has no equivalent in the database. Its conservation and the identification of a mutation in it (ksr^(S-548)) indicate that it plays a role in Ksr function. CA2 is a proline-rich stretch followed by basic residues which may correspond to a class II SH3-domain binding site (PXXPXR/K; Alexandropoulos et al., 1995), although the two fly sequences diverge from the consensus by one amino acid. CA3 is a cysteine-rich domain similar to the one found in other signaling molecules, such as the CR1 domain of Raf. Finally, CA4 is rich in serines and threonines and also contains a MAPK consensus phosphorylation site.

A search of current databases indicated that the Raf kinase members are the closest relatives to the Ksr kinases based on sequence similarity within the kinase domain (e.g. 42% identity/61% similarity between the Dm Ksr and Raf kinase domains) and shared structural features in the N-terminal region (Table 1). Both the Raf and Ksr kinases have a related C-terminal 300 amino acid kinase domain, named CA5 and CR3, respectively (CR3; Heidecker et al., 1992). The spacing and sizes of the domains of the Ksr kinases are well conserved, except for the presence of an additional ˜100 amino acids between the CA4 and CA5 domains of the Drosophila sequences. In addition, they both have a long N-terminal region that contains a cysteine-rich stretch followed by a serine/threonine-rich region, named CA3 and CA4 for Ksr kinases and CR1 and CR2 for Raf kinases. Ksr and Raf kinases also have distinctive features. For instance, the CA1 and CA2 regions found in Ksr kinases are absent from Raf kinases. The Ras-binding domain (RBD) found in the CR1 domain of Raf kinases (Nassar et al., 1995) is absent from Ksr kinases, which suggests that they are regulated differently. Moreover, interaction assays using the yeast two-hybrid system or bacterially-expressed fusion proteins, did not detect any interaction between Ras1 and Ksr, while similar experiments detected an interaction between Ras1 and the CR1 domain of D-Raf. Finally, amino acids in kinase sub-domain VIII, which are important for substrate recognition, are not conserved between Ksr and Raf kinases suggesting that these kinases have different targets. This is supported by the observation that Ksr failed to interact with Dsor1 (D-MEK) in a yeast two-hybrid assay, whereas, D-Raf and Dsor1 interacted strongly.

Ksr functions in multiple RTK pathways.

Recent evidence suggests that RTKs use a similar set of proteins to transduce their signals to the nucleus (see Background). Several lines of genetic evidence suggest that the Ksr kinase corresponds to a new component of this widely used signal transduction pathway. For instance, adult flies homozygous for the sub-viable allele ksr^(S-548) have rough eyes in which ommatidia are missing both outer (R1-R6) and R7 photoreceptor cells. This suggests that, like Ras1 (Simon et al., 1991), ksr has a broader role than just specification of the R7 cell fate. Using the FLP/FRT system (Xu and Rubin, 1993), we did not recover homozygous mutant tissue for the strong allele ksr^(S-638), which indicates that Ksr is required for cell proliferation or survival. In addition, except for the ksr^(S-548) allele, all ksr alleles are recessive lethal and in most cases they die as third instar larvae and lack imaginal discs. This phenotype is often seen with mutations in genes required for cell proliferation (Gatti and Baker, 1989). RNA in situ hybridization showed that ksr MRNA is ubiquitously distributed and is present throughout embryogenesis, consistent with a general role for this kinase.

We directly tested whether ksr is an essential component of the Torso RTK pathway, another Drosophila RTK-dependent signal transduction cascade (reviewed in Duffy and Perrimon, 1994). Torso initiates a signal transduction cascade required for development of the anterior and posterior extremities of the embryo. As for the Sevenless RTK pathway, genetic screens aimed at elucidating this pathway have led to the identification of drk, sos, Ras1 and genes encoding the downstream cassette of kinases (Raf/MEK/MAPK) as being critical for signal propagation (reviewed in Duffy and Perrimon, 1994). This signal transduction cascade appears to control the expression pattern of two genes, tailless (tll) and huckebein (hkb) at the embryonic termini (reviewed in Duffy and Perrimon, 1994). During the cellular blastoderm stage, the posterior domain of expression of both factors depends uniquely on Torso-mediated signaling thereby providing excellent markers of Torso activity.

Embryos derived from mothers homozygous for a torso null mutation have defective termini. The posterior end is missing all structures beyond the seventh abdominal segment, while the anterior end exhibits severe head skeleton defects (reviewed in Duffy and Perrimon, 1994). Consistent with these abnormalities, aberrant expression patterns are observed for tll and hkb; that is, no tll or hkb expression is detected at the posterior end, while tll expression pattern is extended and hkb is retracted at the anterior end. Embryos derived from germlines homozygous for loss-of-function mutations in general RTK components like drk, sos, Ras1 or D-Raf show similar terminal defects, albeit to various degrees, consistent with their role in Torso RTK-mediated signaling (Hou et al., 1995).

To determine whether ksr acts in the Torso pathway, we used the FLP-FDS system (Hou et al., 1995) to generate ksr germline clones and examined the terminal structures of embryos derived from homozygous mutant oocytes. Like embryos derived from Torso mutant mothers, cuticle preparations of ksr^(S-638) embryos revealed severe terminal defects. They are missing posterior structures beyond the seventh abdominal segment and have collapsed head skeletons. In addition, no tll or hkb expression is detected at the posterior end while a broader domain of tll expression and a reduced one for hkb is observed at the anterior extremity. These results indicate that ksr also functions in the Torso pathway, consistent with Ksr being a general component acting downstream of RTKs.

Activated D-Raf rescues terminal defects observed in embryos derived from germlines homozygous for ksr^(S-638).

The inability of ksr mutants to suppress the sE-Raf^(Tor4021) phenotype in the eye suggested that Ksr functions upstream or in parallel to D-Raf, but not downstream. To clarify where ksr functions relative to D-Raf in the Torso pathway, RNA encoding an activated form of D-Raf (Raf^(Tor4021)) was injected into embryos derived from germlines homozygous for ksr^(S-638). If Ksr functions solely upstream of D-Raf then activated D-Raf should rescue the mutant phenotype. In contrast, if Ksr functions solely downstream of D-Raf then injection of activated D-Raf RNA should have no influence on the ksr^(S-638) -associated embryonic phenotype. It is also possible that rescue might be observed if Ksr functions in a pathway parallel to D-Raf and can be bypassed by activation of D-Raf to sufficiently high levels. Injection of activated D-Raf partially rescued the ksr^(S-638) -associated embryonic terminal defects. These results confirm that Ksr does not act downstream of D-Raf.

Experimental Procedures

Fly culture and crosses were performed according to standard procedures. Clonal analysis in the eye was performed on the ksr^(S-638) allele (the strongest suppressor of sev-Ras1^(V12) among the ksr alleles) using the FLP/FRT system (Xu and Rubin, 1993).

ksr^(S-638) germline clones were generated as described in Hou et al. (1995). Cuticle preparation of embryos was performed as described in Belvin et al. (1995). In situ hybridization was performed according to Dougan and DiNardo (1992) using digoxigenin-labelled RNA probes. Injection of embryos was performed as described in Anderson and Nusslein-Volhard (1984). An in vitro trancription kit (Promega) was used to synthesize activated D-Raf RNA from the Raf^(TOR4021) DNA template (Dickson et al., 1992).

Scanning electron microscopy was performed as described by Kimmel et al. (1990). Fixation and sectioning of adult eyes were performed as described by Tomlinson and Ready (1987).

The βGGT-I locus was recovered from a chromosome walk initiated by screening a cosmid library (Tamkun et al., 1992) with a genomic fragment flanking a P-element 1(2)05714! inserted at 25B4-6 (Karpen and Spradling, 1992; Berkeley Drosophila Genome Project, pers. comm.). A 1.7 kb Spe1-Sph1 genomic fragment spanning the S-2126 allele inversion breakpoint was used to screen a Drosophila eye-antennal imaginal disc cDNA library in λgt10. Sixteen related cDNA clones were isolated from ˜700,000 pfu screened.

The ksr gene was isolated from a chromosome walk. Genomic blot analysis of X-ray-induced ksr alleles was performed according to standard procedures (Sambrook et al., 1989). The 2.9 kb and 2.2 kb BamHI fragments from cosmid III identified polymorphisms in the S-69 and S-511 alleles, respectively. A 7 kb EcoRI genomic fragment encompassing all of the 2.9 kb BamHI fragment and part of the 2.2kb BamHI fragment was used along with the 2.2kb BamHI fragment to screen ˜700,000 phage from a Drosophila eye-antennal imaginal disc cDNA library in λgt10. Seven related cDNA clones were isolated and characterized by sequencing.

A D. virilis genomic library was screened at reduced stringency using the Dm Ksr kinase domain as a probe. In brief, filters were hybridized in 5X SSCP; 10X Denhart; 0.1% SDS; 200 μg/ml sonicated salmon sperm DNA at 42° C. for 12 hrs, rinsed several times at room temperature and washed twice for 2 hrs at 50° C. in 1X SSC; 0.1% SDS. 12 genomic clones were identified; one was purified and analyzed by sequencing.

A DNA fragment corresponding to the hb DNA sequence was prepared by PCR from a mouse brain cDNA library and used as a probe to screen a mouse PCC4 teratocarcinoma cDNA library (Stratagene). One full-length cDNA clone, named mKsr- 1, was obtained from 1×10⁶ pfu screened. Using the mKsr-1 kinase domain as a probe, 1×10⁶ pfu of a human fetal brain cDNA library (Clontech) was hybridized at reduced stringency (see above). Thirteen related cDNA clones were isolated and characterized by sequencing. They all represent partial transcripts and only one of them, named hKsr-1, has a complete kinase domain.

DNA sequences were performed by the dideoxy in termination procedure (Sanger et al., 1977) using the Automated Laser Fluorescence (ALF) system (Pharmacia). Templates were prepared by sonicating plasmid DNA and inserting the sonicated DNA into the M13mp10 vector. The entire coding regions of βGGT-I and Ksr cDNAs from each species were sequenced on both strands as well as the genomic regions that correspond to the βGGT-1 and Dm ksr loci. Sequences were analysed using the Staden (R. Staden, MRC of Molecular Biology, Cambridge UK) and the Genetics Computer Group, Inc. software packages. The chromosomal regions for different βGGT-I and ksr mutant alleles were cloned into the λ₋₋ ZAP-express vector (Stratagene) and their respective coding regions were completely sequenced using oligonucleotide primers.

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Dickson, B., et al. (1992), Nature 360, 600-603.

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Duffy, J. B. and Perrimon, N. (1994). Dev. Biol. 166, 380-395.

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Gatti, M. and Baker, B. S. (1989). Genes Dev. 3, 438-453.

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Pharmaceutical lead compound screening assays.

1. Protocol for Ksr--substrate phosphorylation assay.

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

hKsr: 10⁻⁸ -10⁻⁵ M hKsr at 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktail of protease inhibitors.

³² P!γ-ATP 10x stock: 2×10⁻⁵ M cold ATP with 100 μCi ³² P!γ-ATP. Place in the 4° C. microfridge during screening.

Substrate: 2×10⁻⁶ M biotinylated synthetic peptide kinase substrate (MBP, Sigma) at 20 μg/ml in PBS.

Protease inhibitor cocktail (1000X): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVo₃ (Sigma #S-6508) in 10 ml of PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock Neutralite avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 40 μl hKsr (0.1-10 pmoles/40 ul in assay buffer)

Add 10 μl compound or extract.

Shake at 30° C. for 15 minutes.

Add 10 μl ³² P!γ-ATP 10x stock.

Add 10 μl substrate.

Shake at 30° C. for 15 minutes.

Incubate additional 45 minutes at 30° C.

Stop the reaction by washing 4 times with 200 μl PBS.

Add 150 μl scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding (no hKsr added)

b. cold ATP to achieve 80% inhibition.

2. Protocol for hKsr-Raf binding assay.

A. Reagents:

Anti-myc antibody: 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM β-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors.

³³ P hKsr 10x stock: 10⁸ -10⁶ M "cold" hKsr (full length) supplemented with 200,000-250,000 cpm of labeled hKsr (HMK-tagged) (Beckman counter). Place in the 4° C. microfridge during screening.

Protease inhibitor cocktail (1000X): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg Leupeptin (3MB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVo₃ (Sigma #S-6508) in 10 ml of PBS.

Raf: b 10⁻⁸ -10⁻⁵ M myc eptitope-tagged Raf in PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock anti-myc antibody per well overnight at 4° C.

Wash 2X with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2X with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 10 μl compound or extract.

Add 10 μl ³³ P-hKsr (20,000-25,000 cpm/0.1-10 pmoles/well =10⁻⁹ -10⁻⁷ M final concentration).

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25 ° C.

Add 40 μl eptitope-tagged Raf (0.1-10 pmoles/40 ul in assay buffer)

Incubate 1 hour at room temperature.

Stop the reaction by washing 4 times with 200 μl PBS.

Add 150 μl scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding (no hKsr added)

b. Soluble (non-tagged Raf) to achieve 80% inhibition.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 12                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3697 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        GAATTCCAATTATTGCTTTTTCGCATTGCCTAAGCCGTTTAGAGTTGCGGGCGTTAGCGT60                 GCGCGATAGCCGGAGCACCGAACGTCAAGGTCGCTTGGCGAGGGCCACAATGCGGGGCGG120                AGTCCCAGCCATTGGTCCCATCGAATCGTCGAGTCCCCGAGAGGGCGTCTGAAAAAATCA180                ATCGGGCTCCACTCCGTCGCGAATAAGCAGGATGAGCAGCAACAACAACGCACCCGCATC240                GGCTCCAGACACGGGCTCCACCAATGCCAACGATCCCATCTCCGGTTCGCTGTCCGTAGA300                CAGCAACCTGGTTATCATTCAGGACATGATTGATCTCTCGGCCAACCATCTGGAGGGCCT360                GCGAACGCAGTGCGCGATCAGCTCCACGCTGACGCAGCAGGAGATTCGTTGCCTGGAGTC420                GAAGCTGGTGCGATACTTCTCCGAGCTGCTGCTGGCGAAGATGCGGCTAAATGAGCGCAT480                CCCGGCCAACGGGCTTGTGCCCCACACAACGGGCAACGAACTGAGGCAATGGCTGCGCGT540                AGTGGGCCTTAGCCAGGGGACTCTTACCGCCTGCCTTGCTCGCCTGACCACTCTAGAGCA600                AAGCCTGCGTCTCAGCGACGAGGAGATCCGTCAACTCCTGGCTGACAGCCCCAGCCAGCG660                AGAGGAGGAGGAACTGCGACGCCTGACCAGGGCCATGCAGAACTTAAGGAAGTGCATGGA720                GTCGCTGGAGAGCGGTACTGCGGCTAGCAACAACGATCCAGAGCAGTGGCACTGGGACTC780                CTGGGACAGGCCCACCCACATTCATCGCGGCAGTGTGGGAAACATTGGACTGGGTAACAA840                TTCAACCGCCTCCCCGAGAACCCATCATCGCCAGCATGGTGTCAAGGGAAAGAATTCCGC900                TCTGGCCAACTCCACCAACTTCAAAAGTGGCCGCCAATCGCCCTCAGCGACAGAAGAGCT960                GAACAGCACACAGGGTTCCCAGCTGACTTTAACCCTTACGCCCTCGCCACCCAATTCGCC1020               CTTCACGCCTTCCAGTGGGCTGAGCAGCAGCCTTAATGGAACACCACAGAGGAGTCGTGG1080               TACCCCGCCGCCAGCCAGAAAGCACCAGACCTTGCTGAGCCAGAGTCATGTGCAAGTGGA1140               CGGGGAGCAATTAGCCCGCAACCGTTTGCCCACTGATCCCAGCCCCGATAGCCACAGCTC1200               CACCAGCTCGGACATCTTTGTGGACCCAAATACTAATGCCAGCTCCGGAGGAAGTTCCTC1260               GAACGTGCTTATGGTGCCATGCTCTCCGGGCGTGGGTCACGTGGGCATGGGTCATGCAAT1320               CAAGCATCGTTTCACCAAGGCCCTGGGCTTCATGGCCACCTGTACCCTGTGCCAGAAGCA1380               GGTCTTTCACCGCTGGATGAAGTGCACCGACTGCAAGTACATCTGCCACAAGTCATGCGC1440               ACCGCACGTACCGCCCTCCTGTGGACTTCCACGAGAATATGTGGACGAGTTTCGGCACAT1500               AAAGGAGCAGGGAGGATACGCCAGTCTGCCGCATGTGCATGGCGCGGCGAAAGGATCCCC1560               TTTGGTAAAAAAGAGCACCCTGGGTAAGCCCTTGCATCAGCAGCACGGCGATAGCAGTTC1620               GCCGAGTTCCAGCTGCACTAGTTCCACGCCCAGCAGTCCGGCGCTGTTCCAGCAAAGGGA1680               GCGCGAGCTGGATCAGGCGGGCAGCAGCTCTAGCGCCAATCTGTTACCTACGCCTTCGCT1740               TGGCAAGCACCAGCCGAGTCAATTCAACTTTCCCAACGTGACGGTGACGAGCAGTGGCGG1800               AAGCGGTGGTGTATCGCTCATCTCCAATGAACCAGTGCCAGAGCAATTCCCCACGGCGCC1860               TGCAACAGCCAACGGAGGACTTGATAGTCTGGTGAGCAGCTCCAACGGGCACATGAGCTC1920               GCTCATCGGTAGCCAAACTTCAAACGCTTCTACTGCGGCCACCTTGACGGGCAGTCTGGT1980               CAATAGCACAACCACCACCAGCACCTGCAGTTTCTTTCCGCGAAAATTGAGCACAGCCGG2040               TGTGGATAAGAGGACGCCGTTCACCAGCGAGTGCACGGATACCCACAAGTCAAATGACAG2100               CGACAAGACAGTCTCCTTGTCTGGAAGTGCCAGCACGGACTCGGACCGGACACCCGTTCG2160               TGTGGATTCAACGGAAGACGGAGACTCGGGACAATGGCGACAGAACTCGATCTCACTCAA2220               GGAATGGGACATCCCGTATGGTGATCTGCTTCTGCTCGAGCGGATAGGGCAGGGACGCTT2280               CGGCACCGTGCATCGAGCCCTTTGGCACGGAGATGTGGCGGTTAAGCTGCTCAACGAGGA2340               CTATCTGCAAGACGAACACATGCTGGAGACGTTTCGCAGCGAGGTAGCCAACTTCAAGAA2400               CACTCGACACGAGAACCTGGTGCTGTTCATGGGAGCCTGCATGAACCCACCATATTTGGC2460               CATTGTGACTTCATTGTGCAAGGGCAACACCTTGTATACGTATATTCACCAGCGTCGGGA2520               GAAGTTTGCCATGAACCGGACTCTCCTCATTGCCCAGCAGATCGCCCAGGGCATGGGCTA2580               CCTGCACGCAAGGGAGATCATCCACAAAGATCTGCGCACCAAGAACATCTTCATCGAGAA2640               CGGCAAGGTGATTATCACGGACTTTGGGCTGTTCAGCTCCACCAAGCTGCTCTACTGTGA2700               TATGGGCCTAGGAGTGCCCCACAACTGGTTGTGCTACCTGGCGCCGGAGCTAATCCGAGC2760               ATTGCAGCCGGAGAAGCCGCGTGGAGAGTGTCTGGAGTTCACCCCATACTCCGATGTCTA2820               CTCTTTCGGAACCGTTTGGTACGAGCTAATCTGCGGCGAGTTCACATTCAAGGATCAGCC2880               GGCGGAATCGATCATCTGGCAGGTTGGCCGTGGGATGAAGCAGTCGCTGGCCAACCTGCA2940               GTCTGGACGGGATGTCAAGGACTTGCTGATGCTGTGCTGGACCTACGAGAAGGAGCACCG3000               GCCGCAGTTCGCACGCCTGCTCTCCCTGCTGGAGCATCTTCCCAAGAAGCGTCTGGCGCG3060               CAGTCCCTCCCACCCCGTCAACCTTTCCCGTTCCGCCGAGTCCGTGTTCTGAGGGAACTG3120               CAGCATGGCCACTGTCACTGTCTAGTACAATTTCGATCTACCAACTAAGCTAGCTCGCTT3180               TGTGCCCTCGTCCACTCTACACAAACTCTCTCCCAAGGCGAAGTTCTATCGAGCCGAGCG3240               AAGATTGTAAATACATAAACGTAACTACCAAATTATAGCAATCCATTTTAAAAACTACAT3300               ACATATGTGTAGGCATGTATCGGGAGCACTCCAGTTGCAGTTGTTAGCAAACGAAACAAA3360               GGCAAATCAAATGTTAACTCGAAAAAGACAAAACGCTTAAATGTTTAAGAGCAGAGGCAA3420               ACAGAGAAGGCATAGACATACATATACAAACAAACAAACAAGCACTGTGGCAAACATAAA3480               TGTAAACGTTAATCAGGTGAGCAATTTCTAAATTGTTAATTATGTGTAAGAGAACTATAT3540               ATATATATATATATATATATATATATATATATATACATGTATATACAGCAGCAATGTATT3600               GTATATGACGGACTAGTGTTAAATTAAATATATATTGTGAATTATGTATGGTCAAGTGTA3660               TATAGTAAATGGACTTTAAATGCGAAATCGGGAATTC3697                                      (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 966 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetSerSerAsnAsnAsnAlaProAlaSerAlaProAspThrGlySer                               151015                                                                         ThrAsnAlaAsnAspProIleSerGlySerLeuSerValAspSerAsn                               202530                                                                         LeuValIleIleGlnAspMetIleAspLeuSerAlaAsnHisLeuGlu                               354045                                                                         GlyLeuArgThrGlnCysAlaIleSerSerThrLeuThrGlnGlnGlu                               505560                                                                         IleArgCysLeuGluSerLysLeuValArgTyrPheSerGluLeuLeu                               65707580                                                                       LeuAlaLysMetArgLeuAsnGluArgIleProAlaAsnGlyLeuVal                               859095                                                                         ProHisThrThrGlyAsnGluLeuArgGlnTrpLeuArgValValGly                               100105110                                                                      LeuSerGlnGlyThrLeuThrAlaCysLeuAlaArgLeuThrThrLeu                               115120125                                                                      GluGlnSerLeuArgLeuSerAspGluGluIleArgGlnLeuLeuAla                               130135140                                                                      AspSerProSerGlnArgGluGluGluGluLeuArgArgLeuThrArg                               145150155160                                                                   AlaMetGlnAsnLeuArgLysCysMetGluSerLeuGluSerGlyThr                               165170175                                                                      AlaAlaSerAsnAsnAspProGluGlnTrpHisTrpAspSerTrpAsp                               180185190                                                                      ArgProThrHisIleHisArgGlySerValGlyAsnIleGlyLeuGly                               195200205                                                                      AsnAsnSerThrAlaSerProArgThrHisHisArgGlnHisGlyVal                               210215220                                                                      LysGlyLysAsnSerAlaLeuAlaAsnSerThrAsnPheLysSerGly                               225230235240                                                                   ArgGlnSerProSerAlaThrGluGluLeuAsnSerThrGlnGlySer                               245250255                                                                      GlnLeuThrLeuThrLeuThrProSerProProAsnSerProPheThr                               260265270                                                                      ProSerSerGlyLeuSerSerSerLeuAsnGlyThrProGlnArgSer                               275280285                                                                      ArgGlyThrProProProAlaArgLysHisGlnThrLeuLeuSerGln                               290295300                                                                      SerHisValGlnValAspGlyGluGlnLeuAlaArgAsnArgLeuPro                               305310315320                                                                   ThrAspProSerThrAspSerHisSerSerThrSerSerAspIlePhe                               325330335                                                                      ValAspProAsnThrAsnAlaSerSerGlyGlySerSerSerAsnVal                               340345350                                                                      LeuMetValProCysSerProGlyValGlyHisValGlyMetGlyHis                               355360365                                                                      AlaIleLysHisArgPheThrLysAlaLeuGlyPheMetAlaThrCys                               370375380                                                                      ThrLeuCysGlnLysGlnValPheHisArgTrpMetLysCysThrAsp                               385390395400                                                                   CysLysTyrIleCysHisLysSerCysAlaProHisValProProSer                               405410415                                                                      CysGlyLeuProArgGluTyrValAspGluPheArgHisIleLysGlu                               420425430                                                                      GlnGlyGlyTyrAlaSerLeuProHisValHisGlyAlaAlaLysGly                               435440445                                                                      SerProLeuValLysLysSerThrLeuGlyLysProLeuHisGlnGln                               450455460                                                                      HisGlyAspSerSerSerProSerSerSerCysThrSerSerThrPro                               465470475480                                                                   SerSerProAlaLeuPheGlnGlnArgGluArgGluLeuAspGlnAla                               485490495                                                                      GlySerSerSerSerAlaAsnLeuLeuProThrProSerLeuGlyLys                               500505510                                                                      HisGlnProSerGlnPheAsnPheProAsnValThrValThrSerSer                               515520525                                                                      GlyGlySerGlyGlyValSerLeuIleSerAsnGluProValProGlu                               530535540                                                                      GlnPheProThrAlaProAlaThrAlaAsnGlyGlyLeuAspSerLeu                               545550555560                                                                   ValSerSerSerAsnGlyHisMetSerSerLeuIleGlySerGlnThr                               565570575                                                                      SerAsnAlaSerThrAlaAlaThrLeuThrGlySerLeuValAsnSer                               580585590                                                                      ThrThrThrThrSerThrCysSerPhePheProArgLysLeuSerThr                               595600605                                                                      AlaGlyValAspLysArgThrProPheThrSerGluCysThrAspThr                               610615620                                                                      HisLysSerAsnAspSerAspLysThrValSerLeuSerGlySerAla                               625630635640                                                                   SerThrAspSerAspArgThrProValArgValAspSerThrGluAsp                               645650655                                                                      GlyAspSerGlyGlnTrpArgGlnAsnSerIleSerLeuLysGluTrp                               660665670                                                                      AspIleProTyrGlyAspLeuLeuLeuLeuGluArgIleGlyGlnGly                               675680685                                                                      ArgPheGlyThrValHisArgAlaLeuTrpHisGlyAspValAlaVal                               690695700                                                                      LysLeuLeuAsnGluAspTyrLeuGlnAspGluHisMetLeuGluThr                               705710715720                                                                   PheArgSerGluValAlaAsnPheLysAsnThrArgHisGluAsnLeu                               725730735                                                                      ValLeuPheMetGlyAlaCysMetAsnProProTyrLeuAlaIleVal                               740745750                                                                      ThrSerLeuCysLysGlyAsnThrLeuTyrThrTyrIleHisGlnArg                               755760765                                                                      ArgGluLysPheAlaMetAsnArgThrLeuLeuIleAlaGlnGlnIle                               770775780                                                                      AlaGlnGlyMetGlyTyrLeuHisAlaArgGluIleIleHisLysAsp                               785790795800                                                                   LeuArgThrLysAsnIlePheIleGluAsnGlyLysValIleIleThr                               805810815                                                                      AspPheGlyLeuPheSerSerThrLysLeuLeuTyrCysAspMetGly                               820825830                                                                      LeuGlyValProHisAsnTrpLeuCysTyrLeuAlaProGluLeuIle                               835840845                                                                      ArgAlaLeuGlnProGluLysProArgGlyGluCysLeuGluPheThr                               850855860                                                                      ProTyrSerAspValTyrSerPheGlyThrValTrpTyrGluLeuIle                               865870875880                                                                   CysGlyGluPheThrPheLysAspGlnProAlaGluSerIleIleTrp                               885890895                                                                      GlnValGlyArgGlyMetLysGlnSerLeuAlaAsnLeuGlnSerGly                               900905910                                                                      ArgAspValLysAspLeuLeuMetLeuCysTrpThrTyrGluLysGlu                               915920925                                                                      HisArgProGlnPheAlaArgLeuLeuSerLeuLeuGluHisLeuPro                               930935940                                                                      LysLysArgLeuAlaArgSerProSerHisProValAsnLeuSerArg                               945950955960                                                                   SerAlaGluSerValPhe                                                             965                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3681 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        CCCCCAAAAACTATAAAATTTTTCGCGTTTTTCTCATAGCAGAAGCTGTCTCGAAGTCCG60                 CATTTCGCAGGACTGTTCATGTGTGCTTGCAGCAAGCGAAAAAAGCTGGTTGATGTGGAC120                AGAATGTGTGTCAAAGTGGTGCAAACAACAAATGATTTGTAAGTGCGTCTGAAAAAATCA180                ATCAGTTTGTACTGCTGGAAGGGGCGGGCGGGCCACAACAAAATGAGCAGCAGCGCCGCC240                GCCCAGCTGACTGCGCCGCCAGTCAGCAACAGCAACAGCAGCAGCAGTAACAACAATACA300                ACAACGACTGCGAGCGAAAGCAATCTAATCATCATACAGGATATGATTGATCTCTCGGCC360                AACCATCTGGAGGGTCTGCGAACACAGTGCGCAACGAGCGCGACGTTGACGCAACAGGAG420                ATCCGCTGCCTAGAGTCCAAGTTGGTGCGCTACTTCTCCGAACTGCTCTTGACCAAAACG480                AGACTCAACGAACGCATACCCGCGAACGGTCTGCTGCCCCATCATCAGGCTACCGGGAAC540                GAGTTGCGCCAATGGCTGCGAGTAGTTGGACTCAGTCCGGAGTCACTGAATGCATGCCTA600                GCGCGTCTAACGACATTGGAGCAAACACTGCAGCTGAGCGATGAAGAACTGAAACAACTG660                CTTGCCCACAATTCAAGTACCCAGCTGGACGAGGAACTGCGGCGGCTGACCAAAGCGATG720                CATAATCTCCGAAAATGCATGGAAACGCTGGACAGCAGCGGCGCAGTTGCGTCCAACGTC780                GATCCGGAACAATGGCACTGGGACTCCTGGGATCGACCCCATCCGCATCACATGCACCGC840                GGCAGCATTGGCAATATTGGCCTAGGACTAAGCAGCGCCTCACCTCGCGCCCATCATCGT900                CAACATCAACATCAACACGCGAACAGCAAGCCGAAAATTGTTAACAATTCTGCCTCAAGC960                TCCCGCAGCGAACAGCAACCACTGACTGGTTCTCAGTTGACCTTAACACTGACGCCCTCG1020               CCACCCAACTCGCCCTTTACGCCCGCCTCAGGGACGGCATCCGCCAGCGGCACTCCGCAG1080               CGCAGCCGCAGTACCACAACAGCGGCGGGAACGCCACCACCAGCCAAGAAGCATCAAACG1140               CTGCTCATGCACAACAGCAGCGCTTCGGAAACGGCACTCGCGGAGCAGCCTCCACGGCCA1200               CCGCGCAGCCGTCTACCCACAGATCCTAGCCCGGATAGCCACAGCTCGGCCAGCAGTTCG1260               GACATTTTTGTGGACGGTGGCAGTATCAACAGCTCCAATGTACTACTAGTGCCGCCCTCG1320               CCAGGTGTGGCACACGTGGGCATGGGTCATACCATTAAGCACCGTTTCAGTAAATGGTTT1380               GGCTTCATGGCCACGTGCAAACTGTGCCAAAAGCAGATGATGAGCCACTGGTTCAAGTGC1440               ACCGACTGCAAATATATTTGCCACAAGTCCTGTGCGCCGCATGTGCCGCCCTCGTGTGGC1500               CTTCCACCCGAATATGTTCACGAGTTTCGTCAAACTCAGGTGGGCGGCAGATGGGACCCT1560               GCGCAGCACAGCAGCAGCAAGGCATCACCAGTGCCCAGGAAGAGCACGCTGGGCAAACCG1620               CAATTGCAGCAGCCACAGCTGCAGCACGGGGACAGCAGCTCACCAAGCTCGAGCTGCACC1680               AGCTCAACGCCCAGCAGTCCAGCATTGTTCCAGCAGCAGCAACTGCAACTGGCCACGCCC1740               AGCGCCTGCCAGCCGAAACCAGCACCAGCAGCGGTAGCAGCAGCAGCAACACAACAGGGT1800               CAACAGAGTCAATTCAATTTCCCCAACGTGACCATCACAAGCATCAATGCCTGCAATAGT1860               AACGCCAGCGCTGCCCAAACGCTCATATCCAATGAGCCGCAAGCGCATATGGCCACAACG1920               GAGTCCACGCTGACCAATGGCAACAACAACAGCAGCTCCAACAACGGGAGCAGCGCCAAC1980               AACAATAGCAGCAGCAGCAGCAGCTGCTCCAATGGTCACCTGCACTCGCTGACTGGAAGT2040               CAAGTGTCCACGCATTCGGCTACCTCGCAAGTGTCGAATGTCAGTGGCAGCAGCTCGGCC2100               ACCTACACCTCCAGTCTGGTGAACAGCGGCAGTTTCTTTCCGCGGAAATTGAGCAATGCT2160               GGCGTGGACAAGCGGGTGCCCTTTACCAGCGAATATACGGACACGCACAAGTCGAATGAT2220               AGCGACAAGACGGTTTCGTTGTCGGGCAGCGCCAGCACTGACTCGGATCGCACGCCTGTG2280               CGTTTGGACTCCACAGAGGATGGCGACTCGGGCCAATGGCGGCAGAACTCCATATCATTG2340               AAGGAATGGGATATACCCTATGGCGATTTGCACTTGCTGGAGCGCATTGGACAGGGTCGA2400               TTTGGCACCGTGCATCGGGCACTGTGGCATGGCGATGTCGCTGTGAAGCTGCTCAATGAA2460               GACTATCTGCAGGACGAGCACATGCTGGAATCGTTTCGCAACGAGGTGGCCAATTTCAAG2520               AAGACGCGACACGAGAATCTGGTGCTGTTCATGGGCGCCTGCATGAATCCGCCGTATTTG2580               GCCATTGTCACGGCACTATGCAAGGGCAACACCCTGTACACCTATATACATCAGCGAAGG2640               GAGAAGTTTGCAATGAATCGCACGTTGTTGATTGCCCAACAGATTGCCCAGGGCATGGGC2700               TATTTGCATGCCAGGGACATAATACACAAGGATCTGCGCACCAAGAACATTTTTATAGAG2760               AATGGCAAGGTGATCATTACGGACTTTGGCCTATTCAGCTCCACAAAGCTGCTGTACTGT2820               GATATGGGCTTGGGTGTTCCACAAAACTGGCTCTGCTACCTGGCCCCGGAACTAATACGC2880               GCCCTGCAGCCGTGCAAGCCACCCGGCGAGTGTCTAGAGTTCACGTCCTACTCGGATGTT2940               TACTCATTTGGCACCGTTTGGTACGAGCTAATTTGCGGCGAATTCACGTTCAAGGATCAA3000               CCGGCGGAGTCAATCATTTGGCAAGTGGGGCGCGGCATGAAACAGTCGCTGGCCAATCTG3060               CAGTCTGGTCGTGATGTCAAGGACCTGCTGATGCTGTGCTGGACCTATGAAAAGGAGCAC3120               AGGCCGGACTTTGCACGTCTGCTCTCCTTGCTGGAGCATTTGCCAAAGAAGCGCCTGGCA3180               CGCAGTCCCTCGCATCCTGTCAACCTCTCGCGCTCAGCGGAATCTGTATTCTAACCAGCC3240               GATATACAAATATATACGTTTATAGACAAATATGTCATATATGTAAGCAGGCGCGCACAC3300               ACTCACACACACACACACTCTATTTAGCACAATTTCACGTTATATGTAAATGTAAGCTAC3360               ACACATATGCAAACATACGTATGTCACTTTAACTGTAATTGTTGTGCGTGCAAAATGTCA3420               AATGTGAAATTAGCTCTCCGGTAAGGGAAGCAAGAGAATGCGGAGAGCAAAGCTCACTTC3480               CTCAGCCTCATGTATGTGTATGTATGTGTACGACCCTACGACTCTCAAAGAAAAGTTCAA3540               AGTGCATGTGTTACAAAACAAAAAACTGTAAATATACATTTAAAGCAAATGAAACGAAAC3600               TATACATATATGTGTATATCCAATTATAGCAATTTACAAATGCATTGTCAAAATAGTTTT3660               TATCTTTAATTATGTATTGAA3681                                                      (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1003 amino acids                                                   (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        MetSerSerSerAlaAlaAlaGlnLeuThrAlaProProValSerAsn                               151015                                                                         SerAsnSerSerSerSerAsnAsnAsnThrThrThrThrAlaSerGlu                               202530                                                                         SerAsnLeuIleIleIleGlnAspMetIleAspLeuSerAlaAsnHis                               354045                                                                         LeuGluGlyLeuArgThrGlnCysAlaThrSerAlaThrLeuThrGln                               505560                                                                         GlnGluIleArgCysLeuGluSerLysLeuValArgTyrPheSerGlu                               65707580                                                                       LeuLeuLeuThrLysThrArgLeuAsnGluArgIleProAlaAsnGly                               859095                                                                         LeuLeuProHisHisGlnAlaThrGlyAsnGluLeuArgGlnTrpLeu                               100105110                                                                      ArgValValGlyLeuSerProGluSerLeuAsnAlaCysLeuAlaArg                               115120125                                                                      LeuThrThrLeuGluGlnThrLeuGlnLeuSerAspGluGluLeuLys                               130135140                                                                      GlnLeuLeuAlaHisAsnSerSerThrGlnLeuAspGluGluLeuArg                               145150155160                                                                   ArgLeuThrLysAlaMetHisAsnLeuArgLysCysMetGluThrLeu                               165170175                                                                      AspSerSerGlyAlaValAlaSerAsnValAspProGluGlnTrpHis                               180185190                                                                      TrpAspSerTrpAspArgProHisProHisHisMetHisArgGlySer                               195200205                                                                      IleGlyAsnIleGlyLeuGlyLeuSerSerAlaSerProArgAlaHis                               210215220                                                                      HisArgGlnHisGlnHisGlnHisAlaAsnSerLysProLysIleVal                               225230235240                                                                   AsnAsnSerAlaSerSerSerArgSerGluGlnGlnProLeuThrGly                               245250255                                                                      SerGlnLeuThrLeuThrLeuThrProSerProProAsnSerProPhe                               260265270                                                                      ThrProAlaSerGlyThrAlaSerAlaSerGlyThrProGlnArgSer                               275280285                                                                      ArgSerThrThrThrAlaAlaGlyThrProProProAlaLysLysHis                               290295300                                                                      GlnThrLeuLeuMetHisAsnSerSerAlaSerGluThrAlaLeuAla                               305310315320                                                                   GluGlnProProArgProProArgSerArgLeuProThrAspProSer                               325330335                                                                      ProAspSerHisSerSerAlaSerSerSerAspIlePheValAspGly                               340345350                                                                      GlySerIleAsnSerSerAsnValLeuLeuValProProSerProGly                               355360365                                                                      ValAlaHisValGlyMetGlyHisThrIleLysHisArgPheSerLys                               370375380                                                                      TrpPheGlyPheMetAlaThrCysLysLeuCysGlnLysGlnMetMet                               385390395400                                                                   SerHisTrpPheLysCysThrAspCysLysTyrIleCysHisLysSer                               405410415                                                                      CysAlaProHisValProProSerCysGlyLeuProProGluTyrVal                               420425430                                                                      HisGluPheArgGlnThrGlnValGlyGlyArgTrpAspProAlaGln                               435440445                                                                      HisSerSerSerLysAlaSerProValProArgLysSerThrLeuGly                               450455460                                                                      LysProGlnLeuGlnGlnProGlnLeuGlnHisGlyAspSerSerSer                               465470475480                                                                   ProSerSerSerCysThrSerSerThrProSerSerProAlaLeuPhe                               485490495                                                                      GlnGlnGlnGlnLeuGlnLeuAlaThrProSerAlaCysGlnProLys                               500505510                                                                      ProAlaProAlaAlaValAlaAlaAlaAlaThrGlnGlnGlyGlnGln                               515520525                                                                      SerGlnPheAsnPheProAsnValThrIleThrSerIleAsnAlaCys                               530535540                                                                      AsnSerAsnAlaSerAlaAlaGlnThrLeuIleSerAsnGluProGln                               545550555560                                                                   AlaHisMetAlaThrThrGluSerThrLeuThrAsnGlyAsnAsnAsn                               565570575                                                                      SerSerSerAsnAsnGlySerSerAlaAsnAsnAsnSerSerSerSer                               580585590                                                                      SerSerCysSerAsnGlyHisLeuHisSerLeuThrGlySerGlnVal                               595600605                                                                      SerThrHisSerAlaThrSerGlnValSerAsnValSerGlySerSer                               610615620                                                                      SerAlaThrTyrThrSerSerLeuValAsnSerGlySerPhePhePro                               625630635640                                                                   ArgLysLeuSerAsnAlaGlyValAspLysArgValProPheThrSer                               645650655                                                                      GluTyrThrAspThrHisLysSerAsnAspSerAspLysThrValSer                               660665670                                                                      LeuSerGlySerAlaSerThrAspSerAspArgThrProValArgLeu                               675680685                                                                      AspSerThrGluAspGlyAspSerGlyGlnTrpArgGlnAsnSerIle                               690695700                                                                      SerLeuLysGluTrpAspIleProTyrGlyAspLeuHisLeuLeuGlu                               705710715720                                                                   ArgIleGlyGlnGlyArgPheGlyThrValHisArgAlaLeuTrpHis                               725730735                                                                      GlyAspValAlaValLysLeuLeuAsnGluAspTyrLeuGlnAspGlu                               740745750                                                                      HisMetLeuGluSerPheArgAsnGluValAlaAsnPheLysLysThr                               755760765                                                                      ArgHisGluAsnLeuValLeuPheMetGlyAlaCysMetAsnProPro                               770775780                                                                      TyrLeuAlaIleValThrAlaLeuCysLysGlyAsnThrLeuTyrThr                               785790795800                                                                   TyrIleHisGlnArgArgGluLysPheAlaMetAsnArgThrLeuLeu                               805810815                                                                      IleAlaGlnGlnIleAlaGlnGlyMetGlyTyrLeuHisAlaArgAsp                               820825830                                                                      IleIleHisLysAspLeuArgThrLysAsnIlePheIleGluAsnGly                               835840845                                                                      LysValIleIleThrAspPheGlyLeuPheSerSerThrLysLeuLeu                               850855860                                                                      TyrCysAspMetGlyLeuGlyValProGlnAsnTrpLeuCysTyrLeu                               865870875880                                                                   AlaProGluLeuIleArgAlaLeuGlnProCysLysProProGlyGlu                               885890895                                                                      CysLeuGluPheThrSerTyrSerAspValTyrSerPheGlyThrVal                               900905910                                                                      TrpTyrGluLeuIleCysGlyGluPheThrPheLysAspGlnProAla                               915920925                                                                      GluSerIleIleTrpGlnValGlyArgGlyMetLysGlnSerLeuAla                               930935940                                                                      AsnLeuGlnSerGlyArgAspValLysAspLeuLeuMetLeuCysTrp                               945950955960                                                                   ThrTyrGluLysGluHisArgProAspPheAlaArgLeuLeuSerLeu                               965970975                                                                      LeuGluHisLeuProLysLysArgLeuAlaArgSerProSerHisPro                               980985990                                                                      ValAsnLeuSerArgSerAlaGluSerValPhe                                              9951000                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4094 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        GAATTCCCTCGGGGCTTTCCTGCCGAGGCGCCCGTGTCCCCGGGCTCCTCGCCTCGGCCC60                 CCAGCGGCCCCGATGCCGAGGCATGGATAGAGCGGCGTTGCGCGCGGCAGCGATGGGCGA120                GAAAAAGGAGGGCGGCGGCGGGGGCGCCGCGGCGGACGGGGGCGCAGGGGCCGCCGTCAG180                CCGGGCGCTGCAGCAGTGCGGCCAGCTGCAGAAGCTCATCGATATCTCCATCGGCAGTCT240                GCGCGGGCTGCGCACCAAGTGCTCAGTGTCTAACGACCTCACACAGCAGGAGATCCGGAC300                CCTAGAGGCAAAGCTGGTGAAATACATTTGCAAGCAGCAGCAGAGCAAGCTTAGTGTGAC360                CCCAAGCGACAGGACCGCCGAGCTCAACAGCTACCCACGCTTCAGTGACTGGCTGTACAT420                CTTCAACGTGAGGCCTGAGGTGGTGCAGGAGATCCCCCAAGAGCTCACACTGGATGCTCT480                GCTGGAGATGGACGAGGCCAAAGCCAAGGAGATGCTGCGGCGCTGGGGGGCCAGCACGGA540                GGAGTGCAGCCGCCTACAGCAAGCCCTTACCTGCCTTCGGAAGGTGACTGGCCTGGGAGG600                GGAGCACAAAATGGACTCAGGTTGGAGTTCAACAGATGCTCGAGACAGTAGCTTGGGGCC660                TCCCATGGACATGCTTTCCTCGCTGGGCAGAGCGGGTGCCAGCACTCAGGGACCCCGTTC720                CATCTCCGTGTCCGCCCTGCCTGCCTCAGACTCTCCGGTCCCCGGCCTCAGTGAGGGCCT780                CTCGGACTCCTGTATCCCCTTGCACACCAGCGGCCGGCTGACCCCCCGGGCCCTGCACAG840                CTTCATCACGCCCCCTACCACACCCCAGCTACGACGGCACGCCAAGCTGAAGCCACCAAG900                GACACCCCCACCGCCAAGCCGCAAGGTCTTCCAGCTGCTCCCCAGCTTCCCCACACTCAC960                ACGGAGCAAGTCCCACGAGTCCCAGCTGGGAAACCGAATCGACGACGTCACCCCGATGAA1020               GTTTGAACTCCCTCATGGATCCCCACAGCTGGTACGAAGGGATATCGGGCTCTCGGTGAC1080               GCACAGGTTCTCCACAAAGTCATGGTTGTCACAGGTGTGCAACGTGTGCCAGAAGAGCAT1140               GATTTTTGGCGTGAAGTGCAAACACTGCAGGTTAAAATGCCATAACAAGTGCACAAAGGA1200               AGCTCCCGCCTGCAGGATCACCTTCCTCCCACTGGCCAGGCTTCGGAGGACAGAGTCTGT1260               CCCGTCAGATATCAACAACCCAGTGGACAGAGCAGCAGAGCCCCATTTTGGAACCCTTCC1320               CAAGGCCCTGACAAAGAAGGAGCACCCTCCAGCCATGAACCTGGACTCCAGCAGCAACCC1380               ATCCTCCACCACGTCCTCCACACCCTCATCGCCGGCACCTTTCCTGACCTCATCTAATCC1440               CTCCAGTGCCACCACGCCTCCCAACCCGTCACCTGGCCAGCGGGACAGCAGGTTCAGCTT1500               CCCAGACATTTCAGCCTGTTCTCAGGCAGCCCCGCTGTCCAGCACAGCCGACAGTACACG1560               GCTCGACGACCAGCCCAAAACAGATGTGCTAGGTGTTCACGAAGCAGAGGCTGAGGAGCC1620               TGAGGCTGGCAAGTCAGAGGCAGAGGATGACGAGGAGGATGAGGTGGACGACCTCCCCAG1680               CTCCCGCCGGCCCTGGAGGGGCCCCATCTCTCGAAAGGCCAGCCAGACCAGCGTTTACCT1740               GCAAGAGTGGGACATCCCCTTTGAACAGGTGGAACTGGGCGAGCCCATTGGACAGGGTCG1800               CTGGGGCCGGGTGCACCGAGGCCGTTGGCATGGCGAGGTGGCCATTCGGCTGCTGGAGAT1860               GGACGGCCACAATCAGGACCACCTGAAGCTGTTCAAGAAAGAGGTGATGAACTACCGGCA1920               GACGCGGCATGAGAACGTGGTGCTCTTCATGGGGGCCTGCATGAACCCACCTCACCTGGC1980               CATTATCACCAGCTTCTGCAAGGGGCGGACATTGCATTCATTCGTGAGGGACCCCAAGAC2040               GTCTCTGGACATCAATAAGACTAGGCAGATCGCCCAGGAGATCATCAAGGGCATGGGTTA2100               TCTTCATGCAAAAGGCATCGTGCACAAGGACCTCAAGTCCAAGAATGTCTTCTATGACAA2160               CGGCAAAGTGGTCATCACAGACTTCGGGCTGTTTGGGATCTCGGGTGTGGTCCGAGAGGA2220               ACGGCGCGAGAACCAACTGAAACTGTCACATGACTGGCTGTGCTACCTGGCCCCCGAGAT2280               CGTACGAGAAATGATCCCGGGGCGGGACGAGGACCAGCTGCCCTTCTCCAAAGCAGCCGA2340               TGTCTATGCATTCGGGACTGTGTGGTATGAACTACAGGCAAGAGACTGGCCCTTTAAGCA2400               CCAGCCTGCTGAGGCCTTGATCTGGCAGATTGGAAGTGGGGAAGGAGTACGGCGCGTCCT2460               GGCATCCGTCAGCCTGGGGAAGGAAGTCGGCGAGATCCTGTCTGCCTGCTGGGCTTTCGA2520               TCTGCAGGAGAGACCCAGCTTCAGCCTGCTGATGGACATGCTGGAGAGGCTGCCCAAGCT2580               GAACCGGCGGCTCTCCCACCCTGGGCACTTTTGGAAGTCGGCTGACATTAACAGCAGCAA2640               AGTCATGCCCCGCTTTGAAAGGTTTGGCCTGGGGACCCTGGAGTCCGGTAATCCAAAGAT2700               GTAGCCAGCCCTGCACGTTCATGCAGAGAGTGTCTTCCTTTCGAAAACATGATCACGAAA2760               CATGCAGACCACCACCTCAAGGAATCAGAAGCATTGCATCCCAAGCTGCGGACTGGGAGC2820               GTGTCTCCTCCCTAAAGGACGTGCGTGCGTGCGTGCGTGCGTGCGTGCGTGCGTGCGTCA2880               CCAAGGTGTGTGGAGCTCAGGATCGCAGCCATACACGCAACTCCAGATGATACCACTACC2940               GCCAGTGTTTACACAGAGGTTTCTGCCTGGCAAGCTTGGTATTTTACAGTAGGTGAAGAT3000               CATTCTGCAGAAGGGTGCTGGCACAGTGGAGCAGCACGGATGTCCCCAGCCCCCGTTCTG3060               GAAGACCCTACAGCTGTGAGAGGCCCAGGGTTGAGCCAGATGAAAGAAAAGCTGCGTGGG3120               TGTGGGCTGTACCCGGAAAAGGGCAGGTGGCAGGAGGTTTGCCTTGGCCTGTGCTTGGGC3180               CGAGAACCACACTAAGGAGCAGCAGCCTGAGTTAGGAATCTATCTGGATTACGGGGATCA3240               GAGTTCCTGGAGAGTGGACTCAGTTTCTGCTCTGATCCAGGCCTGTTGTGCTTTTTTTTT3300               TTCCCCCTTAAAAAAAAAAAAGTACAGACAGAATCTCAGCGGCTTCTAGACTGATCTGAT3360               GGATCTTAGCCCGGCTTCTACTGCGGGGGGGAGGGGGGGAGGGATAGCCACATATCTGTG3420               GAGACACCCACTTCTTTATCTGAGGCCTCCAGGTAGGCACAAAGGCTGTGGAACTCAGCC3480               TCTATCATCAGACACCCCCCCCCAATGCCTCATTGACCCCCTTCCCCCAGAGCCAAGGGC3540               TAGCCCATCGGGTGTGTGTACAGTAAGTTCTTGGTGAAGGAGAACAGGGACGTTGGCAGA3600               AGCAGTTTGCAGTGGCCCTAGCATCTTAAAACCCATTGTCTGTCACACCAGAAGGTTCTA3660               GACCTACCACCACTTCCCTTCCCCATCTCATGGAAACCTTTTAGCCCATTCTGACCCCTG3720               TGTGTGCTCTGAGCTCAGATCGGGTTATGAGACCGCCCAGGCACATCAGTCAGGGAGGCT3780               CTGATGTGAGCCGCAGACCTCTGTGTTCATTCCTATGAGCTGGAGGGGCTGGACTGGGTG3840               GGGTCAGATGTGCTTGGCAGGAACTGTCAGCTGCTGAGCAGGGTGGTCCCTGAGCGGAGG3900               ATAAGCAGCATCAGACTCCACAACCAGAGGAAGAAAGAAATGGGGATGGAGCGGAGACCC3960               ACGGGCTGAGTCCCGCTGTGGAGTGGCCTTGCAGCTCCCTCTCAGTTAAAACTCCCAGTA4020               AAGCCACAGTTCTCCGAGCACCCAAGTCTGCTCCAGCCGTCTCTTAAAACAGGCCACTCT4080               CTGAGAAGGAATTC4094                                                             (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 873 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        MetAspArgAlaAlaLeuArgAlaAlaAlaMetGlyGluLysLysGlu                               151015                                                                         GlyGlyGlyGlyGlyAlaAlaAlaAspGlyGlyAlaGlyAlaAlaVal                               202530                                                                         SerArgAlaLeuGlnGlnCysGlyGlnLeuGlnLysLeuIleAspIle                               354045                                                                         SerIleGlySerLeuArgGlyLeuArgThrLysCysSerValSerAsn                               505560                                                                         AspLeuThrGlnGlnGluIleArgThrLeuGluAlaLysLeuValLys                               65707580                                                                       TyrIleCysLysGlnGlnGlnSerLysLeuSerValThrProSerAsp                               859095                                                                         ArgThrAlaGluLeuAsnSerTyrProArgPheSerAspTrpLeuTyr                               100105110                                                                      IlePheAsnValArgProGluValValGlnGluIleProGlnGluLeu                               115120125                                                                      ThrLeuAspAlaLeuLeuGluMetAspGluAlaLysAlaLysGluMet                               130135140                                                                      LeuArgArgTrpGlyAlaSerThrGluGluCysSerArgLeuGlnGln                               145150155160                                                                   AlaLeuThrCysLeuArgLysValThrGlyLeuGlyGlyGluHisLys                               165170175                                                                      MetAspSerGlyTrpSerSerThrAspAlaArgAspSerSerLeuGly                               180185190                                                                      ProProMetAspMetLeuSerSerLeuGlyArgAlaGlyAlaSerThr                               195200205                                                                      GlnGlyProArgSerIleSerValSerAlaLeuProAlaSerAspSer                               210215220                                                                      ProValProGlyLeuSerGluGlyLeuSerAspSerCysIleProLeu                               225230235240                                                                   HisThrSerGlyArgLeuThrProArgAlaLeuHisSerPheIleThr                               245250255                                                                      ProProThrThrProGlnLeuArgArgHisAlaLysLeuLysProPro                               260265270                                                                      ArgThrProProProProSerArgLysValPheGlnLeuLeuProSer                               275280285                                                                      PheProThrLeuThrArgSerLysSerHisGluSerGlnLeuGlyAsn                               290295300                                                                      ArgIleAspAspValThrProMetLysPheGluLeuProHisGlySer                               305310315320                                                                   ProGlnLeuValArgArgAspIleGlyLeuSerValThrHisArgPhe                               325330335                                                                      SerThrLysSerTrpLeuSerGlnValCysAsnValCysGlnLysSer                               340345350                                                                      MetIlePheGlyValLysCysLysHisCysArgLeuLysCysHisAsn                               355360365                                                                      LysCysThrLysGluAlaProAlaCysArgIleThrPheLeuProLeu                               370375380                                                                      AlaArgLeuArgArgThrGluSerValProSerAspIleAsnAsnPro                               385390395400                                                                   ValAspArgAlaAlaGluProHisPheGlyThrLeuProLysAlaLeu                               405410415                                                                      ThrLysLysGluHisProProAlaMetAsnLeuAspSerSerSerAsn                               420425430                                                                      ProSerSerThrThrSerSerThrProSerSerProAlaProPheLeu                               435440445                                                                      ThrSerSerAsnProSerSerAlaThrThrProProAsnProSerPro                               450455460                                                                      GlyGlnArgAspSerArgPheSerPheProAspIleSerAlaCysSer                               465470475480                                                                   GlnAlaAlaProLeuSerSerThrAlaAspSerThrArgLeuAspAsp                               485490495                                                                      GlnProLysThrAspValLeuGlyValHisGluAlaGluAlaGluGlu                               500505510                                                                      ProGluAlaGlyLysSerGluAlaGluAspAspGluGluAspGluVal                               515520525                                                                      AspAspLeuProSerSerArgArgProTrpArgGlyProIleSerArg                               530535540                                                                      LysAlaSerGlnThrSerValTyrLeuGlnGluTrpAspIleProPhe                               545550555560                                                                   GluGlnValGluLeuGlyGluProIleGlyGlnGlyArgTrpGlyArg                               565570575                                                                      ValHisArgGlyArgTrpHisGlyGluValAlaIleArgLeuLeuGlu                               580585590                                                                      MetAspGlyHisAsnGlnAspHisLeuLysLeuPheLysLysGluVal                               595600605                                                                      MetAsnTyrArgGlnThrArgHisGluAsnValValLeuPheMetGly                               610615620                                                                      AlaCysMetAsnProProHisLeuAlaIleIleThrSerPheCysLys                               625630635640                                                                   GlyArgThrLeuHisSerPheValArgAspProLysThrSerLeuAsp                               645650655                                                                      IleAsnLysThrArgGlnIleAlaGlnGluIleIleLysGlyMetGly                               660665670                                                                      TyrLeuHisAlaLysGlyIleValHisLysAspLeuLysSerLysAsn                               675680685                                                                      ValPheTyrAspAsnGlyLysValValIleThrAspPheGlyLeuPhe                               690695700                                                                      GlyIleSerGlyValValArgGluGluArgArgGluAsnGlnLeuLys                               705710715720                                                                   LeuSerHisAspTrpLeuCysTyrLeuAlaProGluIleValArgGlu                               725730735                                                                      MetIleProGlyArgAspGluAspGlnLeuProPheSerLysAlaAla                               740745750                                                                      AspValTyrAlaPheGlyThrValTrpTyrGluLeuGlnAlaArgAsp                               755760765                                                                      TrpProPheLysHisGlnProAlaGluAlaLeuIleTrpGlnIleGly                               770775780                                                                      SerGlyGluGlyValArgArgValLeuAlaSerValSerLeuGlyLys                               785790795800                                                                   GluValGlyGluIleLeuSerAlaCysTrpAlaPheAspLeuGlnGlu                               805810815                                                                      ArgProSerPheSerLeuLeuMetAspMetLeuGluArgLeuProLys                               820825830                                                                      LeuAsnArgArgLeuSerHisProGlyHisPheTrpLysSerAlaAsp                               835840845                                                                      IleAsnSerSerLysValMetProArgPheGluArgPheGlyLeuGly                               850855860                                                                      ThrLeuGluSerGlyAsnProLysMet                                                    865870                                                                         (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2846 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        AGAGCAGCGCTGCGCTCGGCCGCGTTGGGAGAGAAGAAGGAGGGCGGTGGCGGGGGTGAC60                 GCGGCTATCGCGGAGGGAGGTGCAGGGGCCGCGGCCAGCCGGACACTGCAGCAGTGCGGG120                CAGCTGCAGAAGCTCATCGACATCTCCATCGGCAGCCTGCGCGGGCTGCGCACCAAGTGC180                GTGGTGTCCAACGACCTCACCCAGCAGGAGATACGGACCCTGGAGGCGAAGCTGGTCCGT240                TACATTTGTAAGCAGAGGCAGTGCAAGCTGAGCGTGGCTCCCGGTGAGAGGACCCCAGAG300                CTCAACAGCTACCCCCGCTTCAGCGACTGGCTGTACACTTTCAACGTGAGGCCGGAGGTG360                GTGCAGGAGATCCCCCGAGACCTCACGCTGGATGCCCTGCTGGAGATGAATGAGGCCAAG420                GTGAAGGAGACGCTGCGGCGCTGTGGGGCCAGCGGGGATGAGTGTGGCCGTCTGCAGTAT480                GCCCTCACCTGCCTGCGGAAGGTGACAGGCCTGGGAGGGGAGCACAAGGAGGACTCCAGT540                TGGAGTTCATTGGATGCGCGGCGGGAAAGTGGCTCAGGGCCTTCCACGGACACCCTCTCA600                GCAGCCAGCCTGCCCTGGCCCCCAGGGAGCTCCCAGCTGGGCAGAGCAGGCAACAGCGCC660                CAGGGCCCACGCTCCATCTCCGTGTCAGCTCTTCCCGCCTCAGACTCCCCCACCCCCAGC720                TTCAGTGAGGGCCTCTCAGACACCTGTATTCCCCTGCACGCCAGCGGCCGGCTGACCCCC780                CGTGCCCTGCACAGCTTCATCACCCCGCCCACCACACCCCAGCTGCGACGGCACACCAAG840                CTGAAGCCACCACGGACGCCCCCCCCACCCAGCCGCAAGGTCTTCCAGCTGCTGCCCAGC900                TTCCCCACACTCACCCGGAGCAAGTCCCATGAGTCTCAGCTGGGGAACCGCATTGATGAC960                GTCTCCTCGATGAGGTTTGATCTCTCGCATGGATCCCCACAGATGGTACGGAGGGATATC1020               GGGCTGTCGGTGACGCACAGGTTCTCCACCAAGTCCTGGCTGTCGCAGGTCTGCCACGTG1080               TGCCAGAAGAGCATGATATTTGGAGTGAAGTGCAAGCATTGCAGGTTGAAGTGTCACAAC1140               AAATGTACCAAAGAAGCCCCTGCCTGTAGAATATCCTTCCTGCCACTAACTCGGCTTCGG1200               AGGACAGAATCTGTCCCCTCGGACATCAACAACCCGGTGGACAGAGCAGCCGAACCCCAT1260               TTTGGAACCCTCCCCAAAGCACTGACAAAGAAGGAGCACCCTCCGGCCATGAATCACCTG1320               GACTCCAGCAGCAACCCTTCCTCCACCACCTCCTCCACACCCTCCTCACCGGCGCCCTTC1380               CCGACATCATCCAACCCATCCAGCGCCACCACGCCCCCCAACCCCTCACCTGGCCAGCGG1440               GACAGCAGGTTCAACTTCCCAGCTGCCTACTTCATTCATCATAGACAGCAGTTTATCTTT1500               CCAGACATTTCAGCCTTTGCACACGCAGCCCCGCTCCCTGAAGCTGCCGACGGTACCCGG1560               CTCGATGACCAGCCGAAAGCAGATGTGTTGGAAGCTCACGAAGCGGAGGCTGAGGAGCCA1620               GAGGCTGGCAAGTCAGAGGCAGAAGACGATGAGGACGAGGTGGACGACTTGCCGAGCTCT1680               CGCCGGCCCTGGCGGGGCCCCATCTCTCGCAAGGCCAGCCAGACCAGCGTGTACCTGCAG1740               GAGTGGGACATCCCCTTCGAGCAGGTAGAGCTGGGCGAGCCCATCGGGCAGGGCCGCTGG1800               GGCCGGGTGCACCGCGGCCGCTGGCATGGCGAGGTGGCCATTCGCCTGCTGGAGATGGAC1860               GGCCACAACCAGGACCACCTGAAGCTCTTCAAGAAAGAGGTGATGAACTACCGGCAGACG1920               CGGCATGAGAACGTGGTGCTCTTCATGGGGGCCTGCATGAACCCGCCCCACCTGGCCATT1980               ATCACCAGCTTCTGCAAGGGGCGGACGTTGCACTCGTTTGTGAGGGACCCCAAGACGTCT2040               CTGGACATCAACAAGACGAGGCAAATCGCTCAGGAGATCATCAAGGGCATGGGATATCTT2100               CATGCCAAGGGCATCGTACACAAAGATCTCAAATCTAAGAACGTCTTCTATGACAACGGC2160               AAGGTGGTCATCACAGACTTCGGGCTGTTTGGGATCTCAGGCGTGGTCCGAGAGGGACGG2220               CGTGAGAACCAGCTAAAGCTGTCCCACGACTGGCTGTGCTATCTGGCCCCTGAGATTGTA2280               CGCGAGATGACCCCCGGGAAGGACGAGGATCAGCTGCCATTCTCCAAAGCTGCTGATGTC2340               TATGCATTTGGGACTGTTTGGTATGAGCTGCAAGCAAGAGACTGGCCCTTGAAGAACCAG2400               GCTGCAGAGGCATCCATCTGGCAGATTGGAAGCGGGGAAGGAATGAAGCGTGTCCTGACT2460               TCTGTCAGCTTGGGGAAGGAAGTCAGTGAGATCCTGTCGGCCTGCTGGGCTTTCGACCTG2520               CAGGAGAGACCCAGCTTCAGCCTGCTGATGGACATGCTGGAGAAACTTCCCAAGCTGAAC2580               CGGCGGCTCTCCCACCCTGGACACTTCTGGAAGTCAGCTGAGTTGTAGGCCTGGCTGCCT2640               TGCATGCACCAGGGGCTTTCTTCCTCCTAATCAACAACTCAGCACCGTGACTTCTGCTAA2700               AATGCAAAATGAGATGCGGGCACTAACCCAGGGGATGCCACCTCTGCTGCTCCAGTCGTC2760               TCTCTCGAGGCTACTTCTTTTGCTTTGTTTTAAAAACTGGCCCTCTGCCCTCTCCACGTG2820               GCCTGCATATGCCCAAGCCGGAATTC2846                                                 (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 875 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        ArgAlaAlaLeuArgSerAlaAlaLeuGlyGluLysLysGluGlyGly                               151015                                                                         GlyGlyGlyAspAlaAlaIleAlaGluGlyGlyAlaGlyAlaAlaAla                               202530                                                                         SerArgThrLeuGlnGlnCysGlyGlnLeuGlnLysLeuIleAspIle                               354045                                                                         SerIleGlySerLeuArgGlyLeuArgThrLysCysValValSerAsn                               505560                                                                         AspLeuThrGlnGlnGluIleArgThrLeuGluAlaLysLeuValArg                               65707580                                                                       TyrIleCysLysGlnArgGlnCysLysLeuSerValAlaProGlyGlu                               859095                                                                         ArgThrProGluLeuAsnSerTyrProArgPheSerAspTrpLeuTyr                               100105110                                                                      ThrPheAsnValArgProGluValValGlnGluIleProArgAspLeu                               115120125                                                                      ThrLeuAspAlaLeuLeuGluMetAsnGluAlaLysValLysGluThr                               130135140                                                                      LeuArgArgCysGlyAlaSerGlyAspGluCysGlyArgLeuGlnTyr                               145150155160                                                                   AlaLeuThrCysLeuArgLysValThrGlyLeuGlyGlyGluHisLys                               165170175                                                                      GluAspSerSerTrpSerSerLeuAspAlaArgArgGluSerGlySer                               180185190                                                                      GlyProSerThrAspThrLeuSerAlaAlaSerLeuProTrpProPro                               195200205                                                                      GlySerSerGlnLeuGlyArgAlaGlyAsnSerAlaGlnGlyProArg                               210215220                                                                      SerIleSerValSerAlaLeuProAlaSerAspSerProThrProSer                               225230235240                                                                   PheSerGluGlyLeuSerAspThrCysIleProLeuHisAlaSerGly                               245250255                                                                      ArgLeuThrProArgAlaLeuHisSerPheIleThrProProThrThr                               260265270                                                                      ProGlnLeuArgArgHisThrLysLeuLysProProArgThrProPro                               275280285                                                                      ProProSerArgLysValPheGlnLeuLeuProSerPheProThrLeu                               290295300                                                                      ThrArgSerLysSerHisGluSerGlnLeuGlyAsnArgIleAspAsp                               305310315320                                                                   ValSerSerMetArgPheAspLeuSerHisGlySerProGlnMetVal                               325330335                                                                      ArgArgAspIleGlyLeuSerValThrHisArgPheSerThrLysSer                               340345350                                                                      TrpLeuSerGlnValCysHisValCysGlnLysSerMetIlePheGly                               355360365                                                                      ValLysCysLysHisCysArgLeuLysCysHisAsnLysCysThrLys                               370375380                                                                      GluAlaProAlaCysArgIleSerPheLeuProLeuThrArgLeuArg                               385390395400                                                                   ArgThrGluSerValProSerAspIleAsnAsnProValAspArgAla                               405410415                                                                      AlaGluProHisPheGlyThrLeuProLysAlaLeuThrLysLysGlu                               420425430                                                                      HisProProAlaMetAsnHisLeuAspSerSerSerAsnProSerSer                               435440445                                                                      ThrThrSerSerThrProSerSerProAlaProPheProThrSerSer                               450455460                                                                      AsnProSerSerAlaThrThrProProAsnProSerProGlyGlnArg                               465470475480                                                                   AspSerArgPheAsnPheProAlaAlaTyrPheIleHisHisArgGln                               485490495                                                                      GlnPheIlePheProAspIleSerAlaPheAlaHisAlaAlaProLeu                               500505510                                                                      ProGluAlaAlaAspGlyThrArgLeuAspAspGlnProLysAlaAsp                               515520525                                                                      ValLeuGluAlaHisGluAlaGluAlaGluGluProGluAlaGlyLys                               530535540                                                                      SerGluAlaGluAspAspGluAspGluValAspAspLeuProSerSer                               545550555560                                                                   ArgArgProTrpArgGlyProIleSerArgLysAlaSerGlnThrSer                               565570575                                                                      ValTyrLeuGlnGluTrpAspIleProPheGluGlnValGluLeuGly                               580585590                                                                      GluProIleGlyGlnGlyArgTrpGlyArgValHisArgGlyArgTrp                               595600605                                                                      HisGlyGluValAlaIleArgLeuLeuGluMetAspGlyHisAsnGln                               610615620                                                                      AspHisLeuLysLeuPheLysLysGluValMetAsnTyrArgGlnThr                               625630635640                                                                   ArgHisGluAsnValValLeuPheMetGlyAlaCysMetAsnProPro                               645650655                                                                      HisLeuAlaIleIleThrSerPheCysLysGlyArgThrLeuHisSer                               660665670                                                                      PheValArgAspProLysThrSerLeuAspIleAsnLysThrArgGln                               675680685                                                                      IleAlaGlnGluIleIleLysGlyMetGlyTyrLeuHisAlaLysGly                               690695700                                                                      IleValHisLysAspLeuLysSerLysAsnValPheTyrAspAsnGly                               705710715720                                                                   LysValValIleThrAspPheGlyLeuPheGlyIleSerGlyValVal                               725730735                                                                      ArgGluGlyArgArgGluAsnGlnLeuLysLeuSerHisAspTrpLeu                               740745750                                                                      CysTyrLeuAlaProGluIleValArgGluMetThrProGlyLysAsp                               755760765                                                                      GluAspGlnLeuProPheSerLysAlaAlaAspValTyrAlaPheGly                               770775780                                                                      ThrValTrpTyrGluLeuGlnAlaArgAspTrpProLeuLysAsnGln                               785790795800                                                                   AlaAlaGluAlaSerIleTrpGlnIleGlySerGlyGluGlyMetLys                               805810815                                                                      ArgValLeuThrSerValSerLeuGlyLysGluValSerGluIleLeu                               820825830                                                                      SerAlaCysTrpAlaPheAspLeuGlnGluArgProSerPheSerLeu                               835840845                                                                      LeuMetAspMetLeuGluLysLeuProLysLeuAsnArgArgLeuSer                               850855860                                                                      HisProGlyHisPheTrpLysSerAlaGluLeu                                              865870875                                                                      (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2126 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        GAATTCCGGCACACATCAGCACTCACACAGCACACAGCACACACACAGCACACATCAGCG60                 CACACACAGCACAGCTTCATCACCCCGCCCACCACACCCCAGCTGCGACGGCACACCAAG120                CTGAAGCCACCACGGACGCCCCCCCCACCCAGCCGCAAGGTCTTCCAGCTGCTGCCCAGC180                TTCCCCACACTCACCCGGAGCAAGTCCCATGAGTCTCAGCTGGGGAACCGCATTGATGAC240                GTCTCCTCGATGAGGTTTGATCTCTCGCATGGATCCCCACAGATGGTACGGAGGGATATC300                GGGCTGTCGGTGACGCACAGGTTCTCCACCAAGTCCTGGCTGTCGCAGGTCTGCCACGTG360                TGCCAGAAGAGCATGATATTTGGAGTGAAGTGCAAGCATTGCAGGTTGAAGTGTCACAAC420                AAATGTACCAAAGAAGCCCCTGCCTGTAGAATATCCTTCCTGCCACTAACTCGGCTTCGG480                AGGACAGAATCTGTCCCCTCGGACATCAACAACCCGGTGGACAGAGCAGCCGAACCCCAT540                TTTGGAACCCTCCCCAAAGCACTGACAAAGAAGGAGCACCCTCCGGCCATGAATCACCTG600                GACTCCAGCAGCAACCCTTCCTCCACCACCTCCTCCACACCCTCCTCACCGGCGCCCTTC660                CCGACATCATCCAACCCATCCAGCGCCACCACGCCCCCCAACCCCTCACCTGGCCAGCGG720                GACAGCAGGTTCAACTTCCCAGCTGCCTACTTCATTCATCATAGACAGCAGTTTATCTTT780                CCAGACATTTCAGCCTTTGCACACGCAGCCCCGCTCCCTGAAGCTGCCGACGGTACCCGG840                CTCGATGACCAGCCGAAAGCAGATGTGTTGGAAGCTCACGAAGCGGAGGCTGAGGAGCCA900                GAGGCTGGCAAGTCAGAGGCAGAAGACGATGAGGACGAGGTGGACGACTTGCCGAGCTCT960                CGCCGGCCCTGGCGGGGCCCCATCTCTCGCAAGGCCAGCCAGACCAGCGTGTACCTGCAG1020               GAGTGGGACATCCCCTTCGAGCAGGTAGAGCTGGGCGAGCCCATCGGGCAGGGCCGCTGG1080               GGCCGGGTGCACCGCGGCCGCTGGCATGGCGAGGTGGCCATTCGCCTGCTGGAGATGGAC1140               GGCCACAACCAGGACCACCTGAAGCTCTTCAAGAAAGAGGTGATGAACTACCGGCAGACG1200               CGGCATGAGAACGTGGTGCTCTTCATGGGGGCCTGCATGAACCCGCCCCACCTGGCCATT1260               ATCACCAGCTTCTGCAAGGGGCGGACGTTGCACTCGTTTGTGAGGGACCCCAAGACGTCT1320               CTGGACATCAACAAGACGAGGCAAATCGCTCAGGAGATCATCAAGGGCATGGGATATCTT1380               CATGCCAAGGGCATCGTACACAAAGATCTCAAATCTAAGAACGTCTTCTATGACAACGGC1440               AAGGTGGTCATCACAGACTTCGGGCTGTTTGGGATCTCAGGCGTGGTCCGAGAGGGACGG1500               CGTGAGAACCAGCTAAAGCTGTCCCACGACTGGCTGTGCTATCTGGCCCCTGAGATTGTA1560               CGCGAGATGACCCCCGGGAAGGACGAGGATCAGCTGCCATTCTCCAAAGCTGCTGATGTC1620               TATGCATTTGGGACTGTTTGGTATGAGCTGCAAGCAAGAGACTGGCCCTTGAAGAACCAG1680               GCTGCAGAGGCATCCATCTGGCAGATTGGAAGCGGGGAAGGAATGAAGCGTGTCCTGACT1740               TCTGTCAGCTTGGGGAAGGAAGTCAGTGAGATCCTGTCGGCCTGCTGGGCTTTCGACCTG1800               CAGGAGAGACCCAGCTTCAGCCTGCTGATGGACATGCTGGAGAAACTTCCCAAGCTGAAC1860               CGGCGGCTCTCCCACCCTGGACACTTCTGGAAGTCAGCTGAGTTGTAGGCCTGGCTGCCT1920               TGCATGCACCAGGGGCTTTCTTCCTCCTAATCAACAACTCAGCACCGTGACTTCTGCTAA1980               AATGCAAAATGAGATGCGGGCACTAACCCAGGGGATGCCACCTCTGCTGCTCCAGTCGTC2040               TCTCTCGAGGCTACTTCTTTTGCTTTGTTTTAAAAACTGGCCCTCTGCCCTCTCCACGTG2100               GCCTGCATATGCCCAAGCCGGAATTC2126                                                 (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 635 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       GluPheArgHisThrSerAlaLeuThrGlnHisThrAlaHisThrGln                               151015                                                                         HisThrSerAlaHisThrGlnHisSerPheIleThrProProThrThr                               202530                                                                         ProGlnLeuArgArgHisThrLysLeuLysProProArgThrProPro                               354045                                                                         ProProSerArgLysValPheGlnLeuLeuProSerPheProThrLeu                               505560                                                                         ThrArgSerLysSerHisGluSerGlnLeuGlyAsnArgIleAspAsp                               65707580                                                                       ValSerSerMetArgPheAspLeuSerHisGlySerProGlnMetVal                               859095                                                                         ArgArgAspIleGlyLeuSerValThrHisArgPheSerThrLysSer                               100105110                                                                      TrpLeuSerGlnValCysHisValCysGlnLysSerMetIlePheGly                               115120125                                                                      ValLysCysLysHisCysArgLeuLysCysHisAsnLysCysThrLys                               130135140                                                                      GluAlaProAlaCysArgIleSerPheLeuProLeuThrArgLeuArg                               145150155160                                                                   ArgThrGluSerValProSerAspIleAsnAsnProValAspArgAla                               165170175                                                                      AlaGluProHisPheGlyThrLeuProLysAlaLeuThrLysLysGlu                               180185190                                                                      HisProProAlaMetAsnHisLeuAspSerSerSerAsnProSerSer                               195200205                                                                      ThrThrSerSerThrProSerSerProAlaProPheProThrSerSer                               210215220                                                                      AsnProSerSerAlaThrThrProProAsnProSerProGlyGlnArg                               225230235240                                                                   AspSerArgPheAsnPheProAlaAlaTyrPheIleHisHisArgGln                               245250255                                                                      GlnPheIlePheProAspIleSerAlaPheAlaHisAlaAlaProLeu                               260265270                                                                      ProGluAlaAlaAspGlyThrArgLeuAspAspGlnProLysAlaAsp                               275280285                                                                      ValLeuGluAlaHisGluAlaGluAlaGluGluProGluAlaGlyLys                               290295300                                                                      SerGluAlaGluAspAspGluAspGluValAspAspLeuProSerSer                               305310315320                                                                   ArgArgProTrpArgGlyProIleSerArgLysAlaSerGlnThrSer                               325330335                                                                      ValTyrLeuGlnGluTrpAspIleProPheGluGlnValGluLeuGly                               340345350                                                                      GluProIleGlyGlnGlyArgTrpGlyArgValHisArgGlyArgTrp                               355360365                                                                      HisGlyGluValAlaIleArgLeuLeuGluMetAspGlyHisAsnGln                               370375380                                                                      AspHisLeuLysLeuPheLysLysGluValMetAsnTyrArgGlnThr                               385390395400                                                                   ArgHisGluAsnValValLeuPheMetGlyAlaCysMetAsnProPro                               405410415                                                                      HisLeuAlaIleIleThrSerPheCysLysGlyArgThrLeuHisSer                               420425430                                                                      PheValArgAspProLysThrSerLeuAspIleAsnLysThrArgGln                               435440445                                                                      IleAlaGlnGluIleIleLysGlyMetGlyTyrLeuHisAlaLysGly                               450455460                                                                      IleValHisLysAspLeuLysSerLysAsnValPheTyrAspAsnGly                               465470475480                                                                   LysValValIleThrAspPheGlyLeuPheGlyIleSerGlyValVal                               485490495                                                                      ArgGluGlyArgArgGluAsnGlnLeuLysLeuSerHisAspTrpLeu                               500505510                                                                      CysTyrLeuAlaProGluIleValArgGluMetThrProGlyLysAsp                               515520525                                                                      GluAspGlnLeuProPheSerLysAlaAlaAspValTyrAlaPheGly                               530535540                                                                      ThrValTrpTyrGluLeuGlnAlaArgAspTrpProLeuLysAsnGln                               545550555560                                                                   AlaAlaGluAlaSerIleTrpGlnIleGlySerGlyGluGlyMetLys                               565570575                                                                      ArgValLeuThrSerValSerLeuGlyLysGluValSerGluIleLeu                               580585590                                                                      SerAlaCysTrpAlaPheAspLeuGlnGluArgProSerPheSerLeu                               595600605                                                                      LeuMetAspMetLeuGluLysLeuProLysLeuAsnArgArgLeuSer                               610615620                                                                      HisProGlyHisPheTrpLysSerAlaGluLeu                                              625630635                                                                      (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 326 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       AspAlaLysSerSerGluGluAsnTrpAsnIleLeuAlaGluGluIle                               151015                                                                         LeuIleGlyProArgIleGlySerGlySerPheGlyThrValTyrArg                               202530                                                                         AlaHisTrpHisGlyProValProValLysThrLeuAsnValLysThr                               354045                                                                         ProSerProAlaGlnLeuGlnAlaPheLysAsnGluValAlaMetLeu                               505560                                                                         LysLysThrArgHisCysAsnIleLeuIlePheMetGlyCysValSer                               65707580                                                                       LysProSerLeuAlaIleValThrGlnTrpCysGluGlySerSerLeu                               859095                                                                         TyrLysHisValHisValSerGluThrLysPheLysLeuAsnThrLeu                               100105110                                                                      IleAspIleGlyArgGlnValAlaGlnGlnMetAspTyrLeuHisAla                               115120125                                                                      LysAsnIleIleHisArgAspLeuLysSerAsnAsnIlePheLeuHis                               130135140                                                                      GluAspLeuSerValLysIleGlyAspPheGlyLeuAlaThrAlaLys                               145150155160                                                                   ThrArgTrpSerGlyGluLysGlnAlaAsnGlnProThrGlySerIle                               165170175                                                                      LeuTrpMetAlaProGluValIleArgMetGlnGluLeuAsnProTyr                               180185190                                                                      SerPheGlnSerAspValTyrAlaPheGlyIleValMetTyrGluLeu                               195200205                                                                      LeuAlaGluCysLeuProTyrGlyHisIleSerAsnLysAspGlnIle                               210215220                                                                      LeuPheMetValGlyArgGlyLeuLeuArgProAspMetSerGlnVal                               225230235240                                                                   ArgSerAspAlaArgArgHisSerLysArgIleAlaGluAspCysIle                               245250255                                                                      LysTyrThrProLysAspArgProLeuPheArgProLeuLeuTrpMet                               260265270                                                                      LeuGluAsnMetLeuArgThrLeuProLysIleHisArgSerAlaSer                               275280285                                                                      GluProAsnLeuThrGlnSerGlnLeuGlnAsnAspGluPheLeuTyr                               290295300                                                                      LeuProSerProLysThrProValAsnPheAsnAsnPheGlnPhePhe                               305310315320                                                                   GlySerAlaGlyAsnIle                                                             325                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 315 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: Not Relevant                                                 (D) TOPOLOGY: Not Relevant                                                     (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       GlyGlnArgAspSerSerTyrTyrTrpGluIleGluAlaSerGluVal                               151015                                                                         MetLeuSerThrArgIleGlySerGlySerPheGlyThrValTyrLys                               202530                                                                         CysLysTrpHisGlyAspValAlaValLysIleLeuLysValValAsp                               354045                                                                         ProThrProGluGlnPheGlnAlaPheArgAsnGluValAlaValLeu                               505560                                                                         ArgLysThrArgHisValAsnIleLeuLeuPheMetGlyTyrMetThr                               65707580                                                                       LysAspAsnLeuAlaIleValThrGlnTrpCysGluGlySerSerLeu                               859095                                                                         TyrLysHisLeuHisValGlnGluThrLysPheGlnMetPheGlnLeu                               100105110                                                                      IleAspIleAlaArgGlnThrAlaGlnGlyMetAspTyrLeuHisAla                               115120125                                                                      LysAsnIleIleHisArgAspMetLysSerAsnAsnIlePheLeuHis                               130135140                                                                      GluGlyLeuThrValLysIleGlyAspPheGlyLeuAlaThrValLys                               145150155160                                                                   SerArgTrpSerGlySerGlnGlnValGluGlnProThrGlySerVal                               165170175                                                                      LeuTrpMetAlaProGluValIleArgMetGlnAspAsnAsnProPhe                               180185190                                                                      SerPheGlnSerAspValTyrSerTyrGlyIleValLeuTyrGluLeu                               195200205                                                                      MetThrGlyGluLeuProTyrSerHisIleAsnAsnArgAspGlnIle                               210215220                                                                      IlePheMetValGlyArgGlyTyrAlaSerProAspLeuSerLysLeu                               225230235240                                                                   TyrLysAsnCysProLysAlaMetLysArgLeuValAlaAspCysVal                               245250255                                                                      LysLysValLysGluGluArgProLeuPheProGlnIleLeuSerSer                               260265270                                                                      IleGluLeuLeuGlnHisSerLeuProLysIleAsnArgSerAlaSer                               275280285                                                                      GluProSerLeuHisArgAlaAlaHisThrGluAspIleAsnAlaCys                               290295300                                                                      ThrLeuThrThrSerProArgLeuProValPhe                                              305310315                                                                      __________________________________________________________________________ 

What is claimed is:
 1. A method of identifying compounds which modulate the binding of a kinase suppressor of ras (Ksr) to a natural intracellular binding target, said method comprising the steps of:forming a mixture comprising:a Ksr, a natural intracellular Ksr binding target, and a candidate agent; incubating said mixture under conditions whereby, but for the presence of said agent, said Ksr selectively binds said binding target at a first binding affinity; detecting a second binding affinity of said Ksr to said binding target, wherein a difference between said first and second binding affinity indicates that said agent modulates the binding of a Ksr to a natural intracellular binding target.
 2. A method according to claim 1, wherein said Ksr binding target comprises a 14-3-3 gene product.
 3. A method according to claim 1, wherein said Ksr binding target comprises a Ksr protein.
 4. A method of identifying agents which modulate the phosphorylation of a substrate by a kinase suppressor of ras (Ksr), said method comprising the steps of:forming a mixture comprising:a Ksr, a Ksr substrate, and a candidate agent; incubating said mixture under conditions whereby, but for the presence of said agent, said Ksr selectively phosphorylates said substrate at a first rate; detecting a second rate of phosphorylation of said substrate by said Ksr, wherein a difference between said first and second rate indicates that said agent modulates the phosphorylation of a Ksr substrate by a Ksr.
 5. A method according to claim 1 wherein said Ksr substrate comprises a 14-3-3 gene product.
 6. A method according to claim 4 wherein said Ksr substrate comprises a Ksr protein. 